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Characterisation of Proteins from Grevillea robusta
and NMR studies of the Serine Protease Inhibitor
Sarah Jane Kruger
B.BioMedSci (Hons)
School of Science
Faculty of Science
Griffith University
Submitted in fulfilment of the requirements
Of the Degree of Doctor of Philosophy
April 2004
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Abstract _____________________________________________________________________________________
Abstract
Proteins that recognise the sugar surface structures on cells have an enormous potential
to be used as tools in the characterisation of these structures. A group of proteins, called
lectins, have been identified that can bind to carbohydrate complexes on the receptors of
cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like
proteins that were different from most other lectins, as they would specifically target the
receptors of white blood cells and not those found on red blood cells. Therefore, the
lectin isolated from G.robusta could be used as a tool to identify the specific surface
structures on white blood cells.
The lectin was isolated using affinity chromatography where a complex
(oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays
confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to
5% of the total protein content) within the seeds of G.robusta. As a result of this low
yield, the identification of the lectin by PAGE was difficult because the levels of protein
were beyond the detection limit of the commercial staining reagents. The lectin was
called the GR2 protein and was characterised as a monocot mannose binding lectin
based on its sugar specificity for only mannose.
A serine protease inhibitor was isolated from the seeds of G.robusta using two different
chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration
chromatography (GR1.GF). Ion exchange chromatography was used to initially
separate the proteins in the crude extract and the fraction containing the GR1 protein
was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing
results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the
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Abstract _____________________________________________________________________________________
extraction process, which lead to the second purification method being established.
Protease inhibitors were added to the buffers prior to being purified by gel filtration
chromatography, which resulted in the GR1 protein being isolated from the crude
extract without the presence of the contaminating protein.
Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da
and the full amino acid sequence was derived by cDNA techniques. Sequence
alignment studies of the GR1 protein showed significant similarities with the Bowman-
Birk inhibitor. The positioning of the cysteine residues were conserved throughout the
Bowman-Birk superfamily, however these residues were not conserved within the GR1
protein. Competitive inhibition assays on the GR1 protein revealed the protein could
inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk
inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-
Birk superfamily of serine protease inhibitors.
The three-dimensional structure of the GR1 protein was determined using two-
dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and
SYBYL® were used to tabulate the information taken from the 2D experiments,
generate structures and minimise these structures respectively. The solution structure of
the GR1 protein was found to contain a region of antiparallel β-sheet structure that
corresponded to the trypsin binding site and the remainder of the protein consisted of
loops and turns that were held together by disulfide bridges (the chymotrypsin-binding
region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor
existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein
is the first member of the Proteaceae family to be characterised as a Bowman-Birk
inhibitor.
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Abstract _____________________________________________________________________________________
This thesis outlines the isolation and biochemical characterisation of the two proteins
found within Grevillea robusta and also describes the steps involved and results
obtained in determining the three-dimensional structure of the GR1 protein.
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Table of Contents _____________________________________________________________________________________
Table of Contents
ABSTRACT............................................................................................................................................... II
TABLE OF CONTENTS.......................................................................................................................... V
LIST OF FIGURES ..............................................................................................................................VIII
LIST OF TABLES ...................................................................................................................................XI
ACKNOWLEDGMENTS ..................................................................................................................... XII
STATEMENT OF ORIGINALITY.....................................................................................................XIII
ABBREVIATIONS ...............................................................................................................................XIV
CHAPTER 1 INTRODUCTION.............................................................................................................. 2
1.1 CARBOHYDRATES AND CELL RECOGNITION................................................................................... 2 1.2 LEUKOCYTE FUNCTION .................................................................................................................. 3 1.3 NEUTROPHIL ANTIGENS ................................................................................................................. 7 1.4 CHARACTERISATION OF NEUTROPHIL GLYCOPROTEINS ............................................................... 10 1.5 CARBOHYDRATE SPECIFICITY OF THE LECTIN ............................................................................... 12 1.6 ANIMAL LECTINS .......................................................................................................................... 15
1.6.1 C-type lectins...................................................................................................................... 15 1.6.2 I-type lectins ....................................................................................................................... 18 1.6.3 Galectins ............................................................................................................................ 19 1.6.4 Pentraxins........................................................................................................................... 21 1.6.5 P-type lectins ...................................................................................................................... 22
1.7 PLANT LECTINS............................................................................................................................. 24 1.7.1 Legume lectins.................................................................................................................... 24 1.7.2 Monocot Mannose binding lectins...................................................................................... 27 1.7.3 Chitin-binding lectins ......................................................................................................... 28 1.7.4 Type II ribosome inactivating protein (RIP) ...................................................................... 29 1.7.5 The Jacalin family .............................................................................................................. 30
1.8 ROLES OF LECTINS IN PLANTS ....................................................................................................... 32 1.9 APPLICATIONS OF PLANT LECTINS ................................................................................................ 33 1.10 SERINE PROTEASE INHIBITORS................................................................................................. 34
1.10.1 Functional Role of Serine Protease Inhibitors ................................................................... 39 1.10.2 Applications of Serine Protease inhibitors......................................................................... 40
1.11 INITIAL RESEARCH................................................................................................................... 41 1.12 AIMS AND EXPECTED OUTCOMES ............................................................................................ 43
CHAPTER 2 EXTRACTION OF PROTEINS FROM GREVILLEA ROBUSTA.............................. 45
2.1 INTRODUCTION ............................................................................................................................. 45 2.2 AMMONIUM SULFATE PRECIPITATION OF CRUDE PROTEINS. ......................................................... 47 2.3 N-TERMINAL SEQUENCING OF THE CRUDE EXTRACT..................................................................... 49 2.4 BIOASSAYS ................................................................................................................................... 50
2.4.1 Agglutination...................................................................................................................... 51 2.4.2 Granulocyte Agglutination test (GAT) ............................................................................... 51 2.4.3 Granulocyte immunofluorescence Test (GIFT) .................................................................. 52 2.4.4 Sugar blocking granulocyte immunofluorescence test ....................................................... 56 2.4.5 Bioassay results for the crude extract of G.robusta ........................................................... 59
2.5 CONCLUSION ................................................................................................................................ 61
CHAPTER 3 PURIFICATION AND CHARACTERISATION OF A LECTIN ISOLATED FROM THE SEEDS OF GREVILLEA ROBUSTA. ........................................................................................... 63
3.1 INTRODUCTION ............................................................................................................................. 63 3.2 PURIFICATION OF THE LECTIN FROM G.ROBUSTA........................................................................... 64 3.3 BIOASSAYS ................................................................................................................................... 67
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3.4 N-TERMINAL SEQUENCING OF THE LECTIN.................................................................................... 70 3.5 CONCLUSION ................................................................................................................................ 72
CHAPTER 4 PURIFICATION OF THE GR1.HPLC PROTEIN ...................................................... 74
4.1 INTRODUCTION ............................................................................................................................. 74 4.1 INITIAL PURIFICATION OF THE PROTEINS FROM THE CRUDE EXTRACT OF G.ROBUSTA .................. 75
4.1.1 Gel Filtration (GF) Chromatography ................................................................................ 75 4.1.2 Ion Exchange (IEX) Chromatography................................................................................ 77
4.2 LARGE SCALE PREPARATION OF THE CRUDE EXTRACT FROM G.ROBUSTA...................................... 83 4.3 FURTHER PURIFICATION OF G.ROBUSTA PROTEINS........................................................................ 90
4.3.1 Further purification of the GR1.Qseph and SO4/5.Qseph proteins. .................................. 91 4.3.2 Further purification of the GR1.HPLC protein.................................................................. 92 4.3.3 Bioassays............................................................................................................................ 95 4.3.4 Purification of the GR1.HighQ protein .............................................................................. 96
4.4 N-TERMINAL SEQUENCING ........................................................................................................... 98 4.5 DETERMINATION OF THE FULL AMINO ACID SEQUENCE AND SEQUENCE ALIGNMENT STUDIES OF THE GR1.HPLC PROTEIN..................................................................................................................... 100 4.6 MASS SPECTROSCOPY (MS) OF THE GR1.HPLC PROTEIN ......................................................... 105 4.7 CONCLUSION .............................................................................................................................. 108
CHAPTER 5 PURIFICATION & CHARACTERISATION OF THE GR1.GF PROTEIN .......... 110
5.1 INTRODUCTION ........................................................................................................................... 110 5.2 EXTRACTION OF THE PROTEINS FROM THE SEEDS OF G.ROBUSTA. ............................................... 111 5.3 PURIFICATION OF THE GR1.GF PROTEIN FROM THE CRUDE EXTRACT ........................................ 112 5.4 BIOASSAYS ................................................................................................................................. 115 5.5 N-TERMINAL SEQUENCING OF THE GR1.GF PROTEIN ................................................................. 117 5.6 MASS SPECTROSCOPY OF THE GR1.GF PROTEIN........................................................................ 118 5.7 SERINE PROTEASE INHIBITION ASSAYS........................................................................................ 119 5.8 CONCLUSION .............................................................................................................................. 121
CHAPTER 6 NMR ASSIGNMENT OF THE GR1 PROTEIN FROM GREVILLEA ROBUSTA. 123
6.1 INTRODUCTION ........................................................................................................................... 123 6.2 NMR SPECTROSCOPY................................................................................................................. 125
6.2.1 One dimensional NMR experiments ................................................................................. 125 6.2.2 Two dimensional NMR experiments ................................................................................. 127
6.2.2.1 Correlated spectroscopy (COSY) and Double quantum filtered COSY (DQF-COSY) ..............129 6.2.2.2 Total correlation spectroscopy (TOCSY)....................................................................................130 6.2.2.3 Nuclear Overhauser Enhancement spectroscopy (NOESY)........................................................131
6.3 THE 1H NMR ASSIGNMENT OF THE GR1 PROTEIN ..................................................................... 133 6.3.1 Solvent suppression .......................................................................................................... 133 6.3.2 Spin system identification................................................................................................. 135 6.3.3 Sequential Assignment of the GR1 protein ....................................................................... 139
6.3.3.1 Sequential assignment of Residues 1-29 .....................................................................................141 6.3.3.2 Sequential assignment of Residues 30-46 ...................................................................................145 6.3.3.3 Sequential assignment of Residues 47-61 ...................................................................................148
6.4 CONCLUSION .............................................................................................................................. 153
CHAPTER 7 STRUCTURAL STUDIES OF THE GR1 PROTEIN................................................. 155
7.1 INTRODUCTION ........................................................................................................................... 155 7.2 SECONDARY STRUCTURE OF THE GR1 PROTEIN ......................................................................... 155
7.2.1 Prediction of secondary structure .................................................................................... 155 7.2.2 Secondary structure assignment....................................................................................... 159
7.3 POSITIONING OF THE DISULFIDE BRIDGES IN THE GR1 PROTEIN ................................................. 161 7.4 THREE DIMENSIONAL STRUCTURE OF THE GR1 PROTEIN............................................................ 164
7.4.1 Structural Restraints......................................................................................................... 164 7.4.2 Structural Calculations .................................................................................................... 165
7.4.2.1 Initial GR1 structures ..................................................................................................................166 7.5 FURTHER REFINEMENT OF THE GR1 PROTEIN............................................................................. 168 7.6 THE OPTIMISED 3D STRUCTURE OF THE GR1 PROTEIN. .............................................................. 174
7.6.1 The structure of section one of the GR1 protein............................................................... 175 7.6.2 The structure of section two of the GR1 protein............................................................... 179 7.6.3 The structure of section three for the GR1 protein........................................................... 183
7.7 CONCLUSION .............................................................................................................................. 186
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CHAPTER 8 CONCLUSIONS............................................................................................................. 190
CHAPTER 9 EXPERIMENTAL ......................................................................................................... 196
9.1.1 Ammonium sulfate precipitation of proteins. ................................................................... 196 9.1.2 Ammonium sulfate precipitation of proteins using protease inhibitors............................ 197
9.2 POLYACRYLAMIDE GEL ELECTROPHORESIS ................................................................................ 197 9.2.1 Sodium Dodecyl Sulfate (SDS) PAGE.............................................................................. 197 9.2.2 Native PAGE .................................................................................................................... 198
9.3 PROTEIN CONCENTRATION ESTIMATION ..................................................................................... 198 9.4 PURIFICATION OF THE GR1 PROTEIN - PART 1............................................................................ 199
9.4.1 Ion exchange chromatography (IEX) ............................................................................... 199 9.4.2 Reverse phase-HPLC ....................................................................................................... 200 9.4.3 High Q chromatography .................................................................................................. 200
9.5 PURIFICATION OF THE GR1 PROTEIN – PART 2 ........................................................................... 200 9.5.1 Gel filtration chromatography ......................................................................................... 200
9.6 PURIFICATION OF A LECTIN FROM G.ROBUSTA............................................................................. 201 9.7 N-TERMINAL SEQUENCING OF PROTEINS ISOLATED FROM G.ROBUSTA ........................................ 201
9.7.1 Deglycosylation of proteins.............................................................................................. 201 9.7.2 Native PAGE and Electroblotting of proteins .................................................................. 202
9.8 BIOASSAYS ................................................................................................................................. 202 9.8.1 Biotinylation of proteins................................................................................................... 202 9.8.2 Granulocyte harvest ......................................................................................................... 203 9.8.3 Granulocyte Agglutination Test (GAT) ............................................................................ 204 9.8.4 Granulocyte Immunofluorescence Test (GIFT)................................................................ 204 9.8.5 Sugar blocking GIFT........................................................................................................ 205
9.9 MASS SPECTROSCOPY................................................................................................................. 206 9.10 NMR SPECTOSCOPY .............................................................................................................. 206
9.10.1 NMR measurements.......................................................................................................... 206 9.10.2 NMR distance restraints................................................................................................... 207 9.10.3 Structure calculations....................................................................................................... 208 9.10.4 Further refinement of the generated structures................................................................ 209
APPENDIX A METHODOLOGY USED TO DETERMINE THE FULL AMINO ACID SEQUENCE OF THE GR1 PROTEIN FROM GREVILLEA ROBUSTA...................................... 211
A-1 RNA EXTRACTION...................................................................................................................... 211 A-2 3’RACE ..................................................................................................................................... 212 A-3 5’RACE ..................................................................................................................................... 214 A-4 SEQUENCING OF THE CDNA ....................................................................................................... 215
APPENDIX B ENZYMATIC INHIBITORY STUDIES OF THE GR1 PROTEIN ISOLATED FROM THE SEEDS OF G.ROBUSTA................................................................................................. 217
B-1 TRYPSIN AND CHYMOTRYPSIN INHIBITION ASSAYS .................................................................... 217
APPENDIX C .............................................................. EXPERIMENTAL RANDOM COIL VALUES 218
APPENDIX D ..................................................................................... STRUCTURE CALCULATIONS 219
D-1 CALIBA AND ANNEAL MACROS USED TO GENERATE THE DYANA STRUCTURES .................. 219 D-2 STEREOCHEMICAL QUALITY OF THE DYANA STRUCTURES ...................................................... 220 D-3 STEREOCHEMICAL QUALITY OF THE SYBYL® MINIMISED STRUCTURES .................................... 221
REFERENCES....................................................................................................................................... 224
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List of Figures _____________________________________________________________________________________
List of Figures
FIGURE 1.1: MAMMALIAN CIRCULATORY CELLS........................................................................................... 4 FIGURE 1.2: (A) P-SELECTIN COMPLEXED WITH SIALYL LEX AND (B) THE SIALYL LEX IN THE BINDING SITE
OF THE SELECTIN. ................................................................................................................................ 5 FIGURE 1.3: SCHEMATIC REPRESENTATION OF LEUKOCYTE TRAFFICKING. ................................................... 6 FIGURE 1.4: THE STRUCTURE OF THE FCγRIIIB. ........................................................................................... 9 FIGURE 1.5: MONOSACCHARIDE STRUCTURE.............................................................................................. 13 FIGURE 1.6: THE ARRANGEMENT OF THE HYDROXYL GROUPS AROUND THE PYRANOSE RING..................... 14 FIGURE 1.7: A SCHEMATIC REPRESENTATION OF THE COLLECTIN PROTEINS. .............................................. 16 FIGURE 1.8: THE STRUCTURE OF THE MBP-A TAKEN FROM THE SIDE (A) AND THE TOP (B). ..................... 17 FIGURE 1.9: STRUCTURAL SIMILARITIES OF THE CRD BETWEEN THE GALECTIN-7 AND LEGUME LECTINS. 21 FIGURE 1.10: THE STRUCTURE OF THE C-REACTIVE PROTEIN (CRP) AND THE SERUM AMYLOID COMPONENT
(SAP). ............................................................................................................................................... 22 FIGURE 1.11: THE STRUCTURES OF LEGUME LECTINS. ................................................................................ 26 FIGURE 1.12: THE STRUCTURE OF THE SNOWDROP LECTIN COMPLEXED WITH METHYL α-D-MANNOSIDE (A)
AND A CLOSE UP OF THE CARBOHYDRATE BINDING SITE (B).............................................................. 27 FIGURE 1.13: THE CRYSTAL STRUCTURE OF THE RICIN A-CHAIN. ............................................................... 30 FIGURE 1.14: THE STRUCTURE OF THE JACALIN LECTIN.............................................................................. 31 FIGURE 1.15: SEQUENTIAL ALIGNMENT OF THE MEMBERS OF THE BOWMAN-BIRK INHIBITOR FAMILY. ..... 36 FIGURE 1.16: THE STRUCTURE OF THE BOWMAN-BIRK INHIBITOR. ............................................................ 39 FIGURE 2.1: GREVILLEA ROBUSTA OR SILKY OAK TREE. .............................................................................. 46 FIGURE 2.2: SDS (A) AND NATIVE (B) PAGE OF THE CRUDE EXTRACT FROM G.ROBUSTA. ....................... 47 FIGURE 2.3: THE BIOTINYLATION OF PROTEINS........................................................................................... 53 FIGURE 2.4: THE SCHEMATIC REPRESENTATION OF THE GIFT BIOASSAY. .................................................. 55 FIGURE 2.5: SCHEMATIC REPRESENTATION OF THE SUGAR BLOCKING GIFT BIOASSAY. ............................ 58 FIGURE 3.1: AFFINITY CHROMATOGRAPHY OF THE CRUDE EXTRACT FROM G.ROBUSTA.............................. 66 FIGURE 3.2: NATIVE PAGE OF THE ELUTED FRACTIONS............................................................................. 67 FIGURE 3.3: N-TERMINAL SEQUENCING RESULTS OF THE LECTIN................................................................ 70 FIGURE 4.1: ELUTED PROTEINS FROM G.ROBUSTA USING GEL FILTRATION CHROMATOGRAPHY.................. 76 FIGURE 4.2: SDS PAGE (A) AND NATIVE PAGE (B) OF ELUTED GF.S200.PK2 FROM GEL FILTRATION
CHROMATOGRAPHY........................................................................................................................... 77 FIGURE 4.3: ANION EXCHANGE CHROMATOGRAPHY OF ELUTED PROTEINS FROM G.ROBUSTA..................... 78 FIGURE 4.4: THE ELUTION PROFILE OF THE CRUDE EXTRACT AFTER MODIFICATIONS TO THE ELUTION
BUFFER AND GRADIENT. .................................................................................................................... 79 FIGURE 4.5: MODIFICATIONS TO THE SALT CONCENTRATION, FLOW RATE AND GRADIENT CONDITIONS..... 80 FIGURE 4.6: NATIVE PAGE OF THE ELUTED PEAKS FROM IEX CHROMATOGRAPHY................................... 81 FIGURE 4.7: ADJUSTMENT OF THE GRADIENT CONDITIONS USING A 10ML Q -SEPHAROSE FF COLUMN. ..... 82 FIGURE 4.8: LARGE SCALE Q-SEPHAROSE COLUMN AT PH 8.0.................................................................... 84 FIGURE 4.9: LARGE SCALE Q-SEPHAROSE COLUMN AT PH 8.5.................................................................... 88 FIGURE 4.10: HYDROXYAPATITE CHROMATOGRAPHY OF THE PROTEINS SO1/2 AND SO4/5 DERIVED FROM
THE Q SEPHAROSE METHOD............................................................................................................... 92 FIGURE 4.11: RP-HPLC OF GR1/4 PROTEINS USING THE GRADIENT 25-32% ACETONITRILE/0.1% TFA. .. 93 FIGURE 4.12: RP-HPLC USING AN ISOCRATIC FLOW RATE AT 29% ACETONITRILE/TFA 0.1%. ................. 94 FIGURE 4.13: THE SEPARATION OF GR1/4.QSEPH PROTEINS USING A HIGH Q COLUMN. ............................ 97 FIGURE 4.14: N-TERMINAL SEQUENCES OF PROTEINS SEEN ON NATIVE PAGE. .......................................... 98 FIGURE 4.15: COMPARISON BETWEEN THE FULL AMINO ACID SEQUENCE AND THE N-TERMINAL
SEQUENCING RESULTS FOR THE GR1 PROTEIN................................................................................. 100 FIGURE 4.16: SEQUENCE ALIGNMENT OF THE GR1 PROTEIN WITH A NUMBER OF PROTEASE INHIBITORS
FROM THE BOWMAN-BIRK PROTEASE INHIBITOR FAMILY................................................................ 102 FIGURE 4.17: POSITION OF THE DISULFIDE BRIDGES OF THE BOWMAN-BIRK INHIBITOR AND SEQUENCE
ALIGNMENT WITH THE GR1 PROTEIN. ............................................................................................. 103 FIGURE 4.18: POSITIVE ELECTROSPRAY OF THE GR1.HPLC PROTEIN. ..................................................... 106 FIGURE 5.1: NATIVE PAGE OF THE CRUDE EXTRACTS PROCESSED WITHOUT AND WITH PROTEASE
INHIBITORS. ..................................................................................................................................... 112 FIGURE 5.2: GEL FILTRATION CHROMATOGRAPHY OF THE CRUDE EXTRACT (CONTAINING PROTEASE
INHIBITORS)..................................................................................................................................... 114 FIGURE 5.3: NATIVE PAGE OF ELUTED FRACTIONS FROM GEL FILTRATION CHROMATOGRAPHY. ............ 114 FIGURE 5.4: N-TERMINAL SEQUENCING HOMOLOGY OF THE ELUTED GR1 PROTEINS. .............................. 117 FIGURE 5.5: MASS SPECTRUM OBTAINED FOR THE GR1.GF PROTEIN. ...................................................... 119 FIGURE 6.1: 1D SPECTRUM OF THE AMIDE REGION FOR THE GR1 PROTEIN............................................... 126
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List of Figures _____________________________________________________________________________________
FIGURE 6.2: THE 1D NMR SPECTRA FOR THE AMIDE REGION OF THE (A) GR1.HPLC AND (B) GR1.GF PROTEINS......................................................................................................................................... 127
FIGURE 6.3: THE SCHEMATIC REPRESENTATION OF THE NOESY EXPERIMENT......................................... 128 FIGURE 6.4: PULSE SEQUENCE FOR THE (A) COSY AND (B) DQF-COSY EXPERIMENTS......................... 129 FIGURE 6.5: THE HN-Hα REGION OF THE DQF-COSY EXPERIMENT FOR THE GR1 PROTEIN. .................. 130 FIGURE 6.6: THE PULSE SEQUENCE FOR THE TOCSY EXPERIMENT. ......................................................... 131 FIGURE 6.7: THE (A) SCHEMATIC PULSE SEQUENCE FOR THE NOESY EXPERIMENT AND (B) THE HN-HN
REGION OF THE NOESY SPECTRA FOR THE GR1 PROTEIN............................................................... 132 FIGURE 6.8: SCHEMATIC REPRESENTATION OF THE WATER SUPPRESSION SEQUENCES FOR (A)
PRESATURATION AND (B) WATERGATE. ..................................................................................... 134 FIGURE 6.9: AN EXAMPLE OF A 2D SPECTRUM.......................................................................................... 136 FIGURE 6.10: A SUMMARY OF THE IDENTIFIED SPIN SYSTEMS IN THE TOCSY SPECTRUM FOR THE GR1
PROTEIN........................................................................................................................................... 138 FIGURE 6.11: SEQUENTIAL ASSIGNMENT OF THE PROTEIN. ....................................................................... 139 FIGURE 6.12: THE TOCSY SPECTRA FOR THE GR1 PROTEIN THAT SHOWS THE REGIONS OF OVERLAPPING
PEAKS. ............................................................................................................................................. 140 FIGURE 6.13: THE FINGERPRINT REGION OF NOESY SPECTRA OF THE GR1 PROTEIN AT 303 K IN 18%
CD3CN/ H2O. ................................................................................................................................ 143 FIGURE 6.14: THE HN-HN REGION OF THE NOESY SPECTRA FOR THE GR1 PROTEIN. ............................ 144 FIGURE 6.15: THE (A) HNI –HNI+1 AND (B) HαI-HNI+1 CONNECTIVITIES FOR RESIDUES 32-46 WITHIN THE
GR1 PROTEIN. ................................................................................................................................. 147 FIGURE 6.16: THE (A) HN-HN AND (B) HαI-HNI+1 CONNECTIVITIES FOR THE RESIDUES 48-61 SEEN IN THE
NOESY SPECTRA............................................................................................................................ 149 FIGURE 7.1: SECONDARY STRUCTURE PREDICTION OF THE GR1 PROTEIN. ............................................... 156 FIGURE 7.2: SECONDARY STRUCTURE PREDICTION COMPARISON BETWEEN THE GR1 PROTEIN (THIS WORK)
AND BOWMAN-BIRK INHIBITOR (WERNER & WEMMER, 1991) FOR THE TRYPSIN-BINDING REGION......................................................................................................................................................... 158
FIGURE 7.3: SECONDARY STRUCTURE PREDICTION COMPARISON BETWEEN THE BOWMAN-BIRK INHIBITOR (WERNER & WEMMER, 1991) AND THE GR1 PROTEIN (THIS WORK) FOR THE CHYMOTRYPSIN-BINDING REGION.............................................................................................................................. 159
FIGURE 7.4: SCHEMATIC REPRESENTATION OF THE HYDROGEN BONDS BETWEEN THE RESIDUES 10-15 AND 19-24............................................................................................................................................... 160
FIGURE 7.5: THE CONSERVATION OF CYSTEINE RESIDUES WITHIN A NUMBER OF BOWMAN-BIRK INHIBITORS AND THE GR1 PROTEIN (THIS WORK). ............................................................................................. 162
FIGURE 7.6: THE NUMBER OF NOE UPPER DISTANCE LIMITS PER RESIDUE IN THE AMINO ACID SEQUENCE OF THE GR1 PROTEIN. .......................................................................................................................... 167
FIGURE 7.7: CONVERGED STRUCTURES OF THE RESIDUES 8-25 OF THE GR1 PROTEIN (A) BEFORE AND (B) AFTER MINIMISATION USING SYBYL®. ........................................................................................... 171
FIGURE 7.8: CONVERGED STRUCTURES OF THE RESIDUES 29-48 OF THE GR1 PROTEIN (A) BEFORE AND (B) AFTER MINIMISATION USING SYBYL®. ........................................................................................... 172
FIGURE 7.9: CONVERGED STRUCTURES FOR THE RESIDUES 50-61 OF THE GR1 PROTEIN (A) BEFORE AND (B) AFTER MINIMISATION USING SYBYL®...................................................................................... 173
FIGURE 7.10: THE SOLUTION STRUCTURE OF THE GR1 PROTEIN............................................................... 174 FIGURE 7.11: CONVERGED STRUCTURES OF THE TRYPSIN-BINDING REGION OF THE GR1 PROTEIN........... 176 FIGURE 7.12: THE SEQUENTIAL ALIGNMENT OF THE TRYPSIN-BINDING REGION OF THE BOWMAN-BIRK
INHIBITOR WITH THE GR1 PROTEIN. ................................................................................................ 177 FIGURE 7.13: STRUCTURAL SIMILARITIES BETWEEN THE (A) GR1 AFTER MINIMISATION AND THE (B)
BOWMAN-BIRK INHIBITOR. ............................................................................................................. 178 FIGURE 7.14: TOP 10 REFINED STRUCTURES FOR RESIDUES 28-48 IN THE GR1 PROTEIN. ......................... 180 FIGURE 7.15: THE SEQUENTIAL SIMILARITIES BETWEEN THE CHYMOTRYPSIN-BINDING REGION OF THE
BOWMAN-BIRK INHIBITOR AND THE GR1 PROTEIN. ........................................................................ 180 FIGURE 7.16: THE CHYMOTRYPSIN-BINDING REGION OF THE (A) BOWMAN-BIRK INHIBITOR AND (B) THE
MINIMISED GR1 PROTEIN. ............................................................................................................... 182 FIGURE 7.17: SEQUENCE ALIGNMENT OF RESIDUES 50-61 FROM THE GR1 PROTEIN WITH THE
CORRESPONDING REGION IN THE BOWMAN-BIRK INHIBITOR. .......................................................... 183 FIGURE 7.18: THE SOLUTION STRUCTURE OF THE FINAL SECTION OF THE GR1 PROTEIN. ......................... 184 FIGURE 7.19: STRUCTURAL DIFFERENCES BETWEEN THE (A) BOWMAN-BIRK INHIBITOR (WERNER &
WEMMER, 1992) AND (B) THE GR1 PROTEIN (THIS WORK)............................................................. 185 FIGURE 7.20: THE SOLUTION STRUCTURE OF THE GR1 PROTEIN............................................................... 187 FIGURE 8.1: SEQUENCE ALIGNMENT OF THE GR1 PROTEIN AND THE BOWMAN-BIRK INHIBITOR. ............ 192 FIGURE A-1: A LIST OF THE DEGENERATIVE PRIMERS DESIGNED FROM THE N-TERMINAL SEQUENCING
RESULTS. ......................................................................................................................................... 213 FIGURE A-2: THE SPECIFIC PRIMERS DEVELOPED FROM THE 3’RACE RESULTS. ...................................... 214
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List of Figures _____________________________________________________________________________________
FIGURE A-3: THE COMPLETE AMINO ACID SEQUENCE OF THE GR1 PROTEIN FROM THE SEEDS OF G.ROBUSTA......................................................................................................................................................... 216
FIGURE B-1: THE INHIBITION CURVES FOR THE GR1 PROTEIN. ................................................................. 217 FIGURE D-1: PROCHECK RESULTS OF THE DYANA GENERATED STRUCTURES..................................... 220 FIGURE D-2: PROCHECK RESULTS OF THE SYBYL® MINIMISED STRUCTURES AFTER (A) 2000 STEPS AND
(B) 4000 STEPS................................................................................................................................ 221 FIGURE D-3: PROCHECK RESULTS AFTER 8000 STEPS OF MINIMISATION USING SYBYL®. ................... 222
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List of Tables _____________________________________________________________________________________
List of Tables
TABLE 1.1: SUMMARY OF THE REVISED NOMENCLATURE OF GRANULOCYTE ALLOANTIGENS.#.................... 8 TABLE 1.2: THE MEMBERS OF THE I-TYPE LECTIN FAMILY.......................................................................... 19 TABLE 1.3: THE MEMBERS OF THE GALECTIN FAMILY.#.............................................................................. 20 TABLE 1.4: MEMBERS OF THE LEGUME LECTIN FAMILY.............................................................................. 25 TABLE 1.5: FAMILIES OF PLANT PROTEIN PROTEASE INHIBITORS. ............................................................... 34 TABLE 1.6: PRELIMINARY BIOLOGICAL CHARACTERISTICS OF THE 8 SPECIES OF PLANTS. .......................... 42 TABLE 2.1: N-TERMINAL SEQUENCES OF THE CRUDE EXTRACT FROM G.ROBUSTA.# .................................... 49 TABLE 2.2: THE GAT AND GIFT RESULTS OF THE CRUDE EXTRACT FROM G.ROBUSTA.# ............................ 59 TABLE 2.3: SUGAR-BLOCKING GIFT RESULTS OF THE CRUDE EXTRACT FROM G.ROBUSTA.# ...................... 60 TABLE 3.1: GAT AND GIFT BIOASSAY RESULTS OF THE PROTEINS ELUTED FROM THE MANNAN-AGAROSE
COLUMN. # ......................................................................................................................................... 68 TABLE 3.2: CONFIRMATION OF THE SUGAR SPECIFICITY OF THE LECTIN.† ................................................... 69 TABLE 4.1: NATIVE PAGE AND GAT BIOASSAY RESULTS OF ELUTED FRACTION FROM THE LARGE-SCALE
PURIFICATION OF PROTEINS FROM G.ROBUSTA SEEDS. # ..................................................................... 85 TABLE 4.2: SATURATION POINT ON GRANULOCYTES USING THE QSEPH.8.5.PK2 FRACTION. # .................... 86 TABLE 4.3: SUGAR-BLOCKING GIFT RESULTS OF QSEPH.8.PK2. †.............................................................. 87 TABLE 4.4: SUMMARY OF NATIVE PAGE AND BIOASSAY RESULTS OF THE PROTEINS ELUTED FROM THE
LARGE-SCALE Q-SEPHAROSE COLUMN AT PH 8.5. #........................................................................... 89 TABLE 4.5: GAT BIOASSAY AND THE BIOLOGICAL EFFECTS OF THE SOLVENTS ON THE CRUDE EXTRACT. # 95 TABLE 4.6: THE GAT AND GIFT BIOASSAY RESULTS OF THE ELUTED FRACTIONS USING HIGH-Q RESIN. # 98 TABLE 5.1: GAT AND GIFT BIOASSAY RESULTS. ..................................................................................... 116 TABLE 6.1: THE SUMMARY OF THE ASSIGNMENT OF THE GR1 PROTEIN.................................................... 150 TABLE 6.2: CHEMICAL SHIFT ASSIGNMENT OF THE GR1 PROTEIN IN 18% CD3CN/ H2O PH 3.5 AT 303 K.
........................................................................................................................................................ 151 TABLE 7.1: SUMMARY OF NMR RESTRAINTS AND STRUCTURAL STATISTICS FROM DYANA FOR ALL 20
STRUCTURES.................................................................................................................................... 168 TABLE 7.2: THE SUMMARY OF THE 20 ENERGY-MINIMIZED NMR STRUCTURES OF THE GR1 PROTEIN
BEFORE AND AFTER SYBYL® MINIMISATION. ................................................................................. 169
___________________________________________________________________ xi
Acknowledgments ____________________________________________________________________________________
Acknowledgments
Firstly, I would like to thank all of my supervisors; Dr Robyn Minchinton, Dr Greg
Pierens and Professor Ron Quinn for giving me the opportunity to undertake this project
and for providing unfailing support and advice when it was greatly needed.
I would like to thank the Australian Postgraduate Award (industry) and Natural
Products Discovery (formally AstraZeneca) for funding me throughout the duration of
the project.
A very special thanks to all of the staff at the Australian Red Cross Blood Service
(ARCBS) and Natural Products Discovery for their continual help, guidance and
support throughout the years. I would like to thank Helen Clague for her contribution to
the project, as without her sequencing results, the NMR assignment would have been
very challenging.
To my family, thank you very much for the support and encouragement through all the
good and the difficult times. I would also like to thank Frank Stevenson and Lyle
McMillen, for reviewing the thesis.
To all my friends, in particular, Jay, Elizabeth, Heather, Matthew, Sylvia, and Rama,
thank you for your support and listening to me over the years. Thank you for not
saying, “Have you finished yet?”
Finally I would like to thank Shane who has put up with me during my self-doubting
stages, and also providing support throughout.
_________________________________________________________________ xii
Statement of Originality ____________________________________________________________________________________
Statement of Originality
This work has not previously been submitted for a degree or diploma in any university.
To the best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made in the thesis
itself.
………………………………
Sarah Jane Kruger
_________________________________________________________________ xiii
Abbreviations ____________________________________________________________________________________
Abbreviations
Ala alanine Arg arginine Asn asparagine Asp aspartic acid BBI Bowman-Birk inhibitor BLAST basic local alignment search tool cDNA copy DNA CD3CN acetonitrile CHT hydroxyapatite COSY correlated spectroscopy CRD carbohydrate recognition domain Cys cysteine DMSO dimethyl-sulfoxide DSS sodium 3-(trimethylsilyl)-1-propanesulfonic acid DQF-COSY double quantum filtered correlated spectroscopy DYANA dynamic algorithm for NMR applications EDTA ethylenediaminetetra acetic acid FID Fourier Induced Decay FITC fluorescein isothiocyanate FT Fourier Transform fuc fucose gal galactose GalNAc N-acetylgalactosamine GAT granulocyte agglutination test GIFT granulocyte immunofluorescence test glc glucose GlcNAc N-acetylglucosamine Gln glutamine Glu glutamic acid Gly glycine His histidine HNA human neutrophil antigen Ile isoleucine lac lactose Leu leucine LMW low molecular weight Lys lysine malt maltose man mannose MBP mannose binding protein MCF mean channel fluorescence Met methionine MWCO molecular weight cut off NaCl sodium chloride NeuNAc N-acetylneuraminic acid NMR nuclear magnetic resonance
_________________________________________________________________ xiv
Abbreviations ____________________________________________________________________________________
NOESY Nuclear Overhauser Enhancement spectroscopy PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PDB protein databank Phe phenylalanine PIC protease inhibitor cocktail PMSF phenylmethylsulfonyl fluoride Pro proline PVDF polyvinylidene difluride rf radio frequency RMSD root mean square deviation SDS sodium dodecyl sulfate Ser serine SiaLex sialyl Lewis x isomer SiaLea sialyl Lewis a isomer TBS Tris buffered saline Thr threonine TOCSY total correlation spectroscopy Trp tryptophan Tyr tyrosine Val valine
_________________________________________________________________ xv
Chapter 1 Introduction _________________________________________________________________________________
Chapter 1 Introduction
_________________________________________________________________ 1
Chapter 1 Introduction _________________________________________________________________________________
Chapter 1 Introduction
1.1 Carbohydrates and Cell Recognition
Cell recognition plays an important role in a number of biological events including
processes such as fertilisation, embryogenesis, cell migration, organ formation, immune
defence and microbial infection (Sharon & Lis, 1989). In order for these processes to
occur, specific interactions between molecules are essential. Emil Fisher in 1897
pioneered the concept that specific molecules interact with each other in a similar way
that a key fits specifically into a lock (Sharon & Lis, 1993). This “ lock and key”
concept is still used today to explain the specific interactions between enzymes and
substrates.
In the 1970’s it was established that almost all cells have carbohydrates on their surfaces
in the form of glycoproteins, glycolipids and polysaccharides. However at this time, the
possibility of carbohydrates on the cell surfaces playing a role in cell recognition was a
very farfetched concept due to the complexity of the surface structures. It was this
complexity which prompted the notion that carbohydrates could encode large amounts
of biological information on their monomer units.
Theoretically, peptides and oligonucleotides could exchange information based on the
number of monomeric units they contain and also their sequence. Carbohydrates on the
other hand, could encode more information due to the position and the configuration (α
or β) of the glycosidic units (Sharon and Lis, 1989). If two molecules of a single amino
acid or nucleotide were taken for example, they could form one dipeptide or one
dinucleotide while 2 molecules of a monosaccharide could form 11 different
_________________________________________________________________ 2
Chapter 1 Introduction _________________________________________________________________________________
disaccharides. Therefore, if 4 different amino acids were taken, only 24 different
tetrapeptides can be formed while 4 different monosaccharides can form a staggering
35560 different tetrasaccharides. It could be proposed that the carbohydrate may be
involved in transfer of biological information from cell to cell via its receptor.
Therefore, the probability of carbohydrates playing a role in the transfer of biological
information was possible.
1.2 Leukocyte Function
The circulatory system of animals is made up of 2 types of cells: red blood cells and
white blood cells. Both of these cells are both derived from the pluripotent stem cell in
the bone marrow. Red blood cells or erythrocytes function by transporting oxygen and
carbon dioxide (bound to haemoglobin) in the blood around the body. The white blood
cells or leukocytes function as the defence system against infection from foreign
material and are able to migrate across the blood vessel walls to the site of infection
(Alberts et. al., 1994).
A number of different types of cells are defined as leukocytes and they include
polymorphonuclear granulocytes, monocytes and lymphocytes (Figure 1.11).
Granulocytes are made up of three different cell types called neutrophils, basophils and
eosinophils where the neutrophils are the most common of the granulocytes and
function in conjunction with the macrophages, to destroy and phagocytise small
microorganisms. Basophils secrete histamine to help in inflammatory reactions and
eosinophils destroy parasites and regulate allergic reaction responses and monocytes
mature into macrophages, which aids in the destruction of foreign matter. Lymphocytes
_________________________________________________________________ 3
Chapter 1 Introduction _________________________________________________________________________________
are divided into either T- or B-lymphocytes, which are commonly referred to as T- and
B-cells (Alberts et. al, 1994).
l
Polymorphonuclear ulocytes Gran
Figure 1.1: Mammalian circulato
The recruitment of the leukocytes to
a group of structurally related lect
lectin family, are defined as C
transmembrane proteins and are lo
1993). These proteins are made up
N-terminus (outside of the cell) a
_____________________________
Pluripotent stem cel
ry c
th
ins
a2+
cate
of
nd
___
Leukocytes
Erythrocytess Lymphocytes
B- & T- lymphocytes
ells.
e site of injury resul
called selectins. S
dependent protein
d on the surface o
a carbohydrate reco
an epidermal grow
_______________
Monocyte
s
neutrophils basophils eosinphiltin
el
s
f
g
th
__
macrophage
g in inflammation involves
ectins belong to the C-type
are classified as type I
cells (Drickamer & Taylor,
nition domain (CRD) at the
factor (EGF)-like domain.
________________ 4
Chapter 1 Introduction _________________________________________________________________________________
This is followed by a series of short complement binding proteins like units, a
membrane spanning region and finally the C-terminus located in the cytoplasm (Weis et
l, 1998, Lis & Sharon, 1998).
1,3-linked fucose to be important for binding to all
., 1999).
mplexed with Sialyl Lex and (B) the Sialyl Lex in the
was taken from the RSCB PDB database
DB ID: 1G1R: Somers et. al., 2000).
a
These selectins are specific for sialyl-Lex and its isomer sialyl-Lea oligosaccharide
structures and this interaction is shown in Figure 1.2. Sialyl Lewisx is a sialyated,
fucocylated tetrasaccharide and is defined by the following structure
NeuNAcα2→3Galβ1→4(Fucα1→3) GlcNAc-. Mutational studies on this
tetrasaccharide have shown the α
selectins (Fukuda et. al
A
B
Figure 1.2: (A) P-selectin co
binding site of the selectin.
The Sialyl Lex is shown in yellow. The structure
(P
_________________________________________________________________ 5
Chapter 1 Introduction _________________________________________________________________________________
Selectins mediate the adhesion of the circulating leukocytes to the endothelial cells,
which leads to the removal and relocation of cells to the site of infection. The
mechanism behind the leukocyte removal involves the specific binding of the selectin
(located on either the leukocyte (L-selectin) or on the endothelium (E- or P- selectin))
to their corresponding receptor (SiaLex and SiaLea). Prior to this binding, the leukocyte
“rolls” along the endothelium, making and breaking bonds as it rolls, resulting in the
slowing down and the eventual binding of the circulating cells to the endothelial
ceptors (Figure 1.3). Once bound, the leukocytes are removed from circulation and
relocated to the site of infection.
re
F
(
o
S
_
A
igure 1.3: Schematic representation of leuk
) Adhesion and migration of cells through the enA
f (B) selectins to lymphocytes and (C) neutrop
pringer 1990.
_____________________________________
B
ocyte trafficking.
dothelial wall. Mechanism for the adhesion
hils to the endothelial cells. Taken from
___________________________ 6
Chapter 1 Introduction _________________________________________________________________________________
P-selectins are found on both the platelets and endothelial cells and are expressed within
a short period of time (minutes) by molecules such as thrombin, histamine, substance-P
and peroxide. L-selectins found on lymphocytes such as T-cells are involved in the
circulation of lymphocytes through the peripheral lymph nodes and their expression
decreases with the introduction of inflammatory mediators such as cytokines. However,
an increase in the level of cytokines can result in the activation of the E-selectins, which
re found on the surface of the endothelium. E-selectins bind to monocytes and
infection (chemotaxis)) and phagocytosis. Degranulation of
reign material using peroxides formed from the neutrophil and the intracellular
destruction of the foreign material are also functions of the neutrophil (De Haas et. al,
1995; Zhou et. al. 1993).
he allele, are also shown in this table (Clay and
a
neutrophils and they are expressed within hours rather than minutes like the P-selectins
(Lasky, 1992).
Glycoproteins and glycolipids found on the surface of neutrophils are involved in a
number of documented neutrophil functions. These functions include the initial
identification of foreign material (which results in the recruitment and migration of
neutrophils to the site of
fo
1.3 Neutrophil Antigens
Like red blood cells, neutrophil surface glycoproteins also contain blood group antigens.
The Granulocyte Antigen and the Australian Society for Blood Transfusion (ASBT)
working parties on platelet and granulocyte serology (1999) have reviewed the
nomenclature of these antigens as shown in Table 1.1. Previous nomenclature of the
neutrophil antigens (NA1, NA2 etc.), the membrane location of the antigen on the
glycoprotein and the location on t
_________________________________________________________________ 7
Chapter 1 Introduction _________________________________________________________________________________
Stroncek, 1994). Some of the previously assigned neutrophil antigens (NB2, 5a, 9a, &
they
have not been fully characterised.
Table 1.1: Summary of the revised nome #
System nym
9b) are not listed in the table, however they do exist but are not included because
nclature of Granulocyte alloantigens.
Antigen Antigen Location Acro Alleles
HNA-1 HNA-1
HNA-1a FcgRIIIb
HNA-2 HNA-2a gp 50-64 NB1 not defined HNA-3 HNA-3a gp 70-95 5b not defined
NA1 FCGR3B*1b FcgRIIIb NA2 FCGR3B*2
HNA-1c FcgRIIIb SH FCGR3B*3
HNA-4 HNA-4a CD11b MART CD11B*1 HNA-5 HNA-5a CD11a OND CD11A*1
# HNA = Human neutrophil antigen, gp = glycoprotein.
The HNA-1a (NA1) and HNA-1b (NA2) antigens seen in Table 1.1 are located on the
FcγRIIIb receptor. The FcγRIIIb is one of two FcγRIII receptors (or CD16), that have a
low affinity for IgG and is found on the surface of neutrophils where it is anchored via a
glycan-phosphatidylinositol moiety (Vossebeld et. al., 1997). The differences between
the HNA-1a and HNA-1b allotypes are the result of 5 nucleotides that correspond to the
tal number of glycosylation sites within the antigen. The HNA-1b antigen contains 6
in calcium influx, the release of
xygen and the phagocytosis of opsonised bacteria or viruses (Moldovan et al., 1999).
The expression of the receptor is lost when the neutrophil is exposed to inflammatory
9).
to
glycosylation sites compared to 4 for HNA-1a (Deo et. al., 1997). The structure of the
FcγRIIIb determined by X-ray crystallography is shown in Figure 1.4.
Neutrophil activation of the FcγRIIIb often results
o
signals such as cytokines (Moldovan et. al., 199
_________________________________________________________________ 8
Chapter 1 Introduction _________________________________________________________________________________
Figure 1.4: The structure of the FCγRIIIb.
The FcγRIIIb is made up of two regions of antiparallel β-sheet structures that contain 2 α-
helices. Structures were taken from the RSCB PDB database (PDB ID: 1E4J, Sondrmann et.
al., 2000).
A number of severe disorders are the result of neutrophil antigens including neonatal
alloimmune neutropenia, immune neutropenia after bone marrow transplantation
(decrease in the circulating white blood cells) (Stroncek et. al, 1994), and severe
transfusion reactions resulting in acute lung injury (Bux, 1996; Popovsky et. al., 1992).
The characterisation of these neutrophil antigens (and the receptors they target) would
be advantageous for the diagnosis and treatment of these disorders.
_________________________________________________________________ 9
Chapter 1 Introduction _________________________________________________________________________________
1.4 Characterisation of Neutrophil Glycoproteins
Protein-carbohydrate interactions are common in nature. Previously, the biochemical
characterisation of neutrophil glycoproteins was completed using murine monoclonal
antibodies. This proved to be very expensive and alternative methods were required.
The discovery of a group of sugar binding proteins (called lectins), which were found to
bind to the neutrophil surface glycoproteins, provided an alternate and more cost
effective method for the study of these surface structures (Minchinton, 1995;
Minchinton et. al., 1997a; Stroncek et. al., 1994; Zhou et. al., 1993).
Lectins were first identified over a century ago in plants where an extract from caster
beans (ricin) was found to agglutinate red blood cells. William Boyd first saw the
discovery of the effects of lectins on different blood groups. He found the extract from
lima beans (Phaseolus limensis) caused agglutination in some individuals but not all
and this lectin extract was found to be specific for blood type A and it also reacted
weakly with blood type B cells (only when concentrated). Powerful agglutinins were
identified and derived from the tufted vetch (Vicca cracca) and were found to have a
stronger specificity for blood type A than for the blood type B or O cells. Boyd
concluded that these proteins were selective in their interactions with the carbohydrates
and cells and that the sugars were immunodeterminates for the specificity of the blood
types (Sharon & Lis, 1995). This “selective” agglutination of red blood cells resulted in
the ABO blood typing system used today.
Since then, lectins have been found in animals, bacteria, viruses and fungi. They are
defined as non-immune proteins that bind reversibly and with high specificity to the
carbohydrate surface structures of the cell (Goldstein et. al, 1980). Each lectin molecule
_________________________________________________________________ 10
Chapter 1 Introduction _________________________________________________________________________________
contains two or more carbohydrate binding sites, which allows the protein to form
cross-links between the cells resulting in agglutination. The exception to this is the
toxin ricin, which only has one binding site. The majority of these lectins were isolated
using affinity chromatography where specific immobilised carbohydrate resins were
used (Rüdiger, 1998).
_________________________________________________________________ 11
Chapter 1 Introduction _________________________________________________________________________________
1.5 Carbohydrate specificity of the lectin
Lectins are defined by their ability to bind reversibly to simple or complex
carbohydrates. They can be specific for a range of different carbohydrates and some
will bind with a higher affinity than others will. Different lectins can be specific for the
same carbohydrate and this specificity is usually for foreign glycans (Peumans & Van
Damme, 1998). The multivalency and the spatial relationship with the binding site of
the lectin and its oligosaccharide mediate the lectin function (Rini, 1995; Weis &
Drickamer, 1996).
Lectins are subdivided into groups that are based on their specificity for
monosaccharides. These monosaccharide groups are the mannose/glucose specific,
galactose/GalNAc specific, GlcNAc specific, fucose specific, sialic acid specific
(Goldstein & Poretz, 1986), monocot mannose binding lectins (Van Damme et. al.,
1995) and the maltose/mannose binding lectins (Peumans et. al., 1997). The structures
of these monosaccharides are shown in Figure 1.5. Lectins that do not fall into any of
these groups are referred as “complex carbohydrates” due to their specificity for
complex carbohydrates (Van Damme et. al., 1998).
_________________________________________________________________ 12
Chapter 1 Introduction _________________________________________________________________________________
)
Fig
All
Van
carb
The
betw
is c
with
___
Mannose (Man)
N-ac
ure 1.5: Monosaccharide stru
sugars are in the D-conformation u
der Waals interactions and h
ohydrate ring has been foun
refore, the positioning of the hy
een the lectin and carbohydrat
hosen by the lectin, the size o
in this site are critical (Drickam
_________________________
Glucose (Glc
etylgalactosamine (GalNAc)
cture.
nless otherwise indicated.
ydrogen bonding with the
d to stabilise the lectin-
droxyl groups, hydrogen bo
e are important. To ensure
f the lectin binding site an
er, 1997).
_______________________
N-acetylglucosamine(GlcNAc)
Galactose (Gal)
N-acetylneuraminic acid(Neu-Nac)
L-fucose (Fuc)oxygen atom within the
carbohydrate interaction.
nds and steric exclusions
the specific carbohydrate
d the number of contacts
______________ 13
Chapter 1 Introduction _________________________________________________________________________________
Structural studies on lectin-carbohydrate interactions have shown the positioning of the
3- and 4- hydroxyl groups within the pyranose ring to be essential for specificity. The
arrangement of the hydroxyl groups for the Man/Glc and GlcNAc lectins were found to
be equatorial at positions 3 & 4 and in the axial conformation at position 4 in the
Gal/GalNAc lectins (Figure 1.6). Therefore, lectins defined as mannose specific will
not bind to the monosaccharide galactose due to the orientation of the hydroxyl group
(Drickamer, 1997).
Figure 1.6: The arrangement of the hydroxyl groups around the pyranose ring.
Lectins that are specific for mannose are usually specific for glucose but are not specific for
galactose due to the positioning of the 4-hydroxyl group (shown in red).
_________________________________________________________________ 14
Chapter 1 Introduction _________________________________________________________________________________
1.6 Animal lectins
A number of lectins have been identified in animals. These lectins are divided into five
groups that are dependent on their structural homologies rather than their carbohydrate
specificities. These groups are the C-type, I-type, galectins (S-type), pentraxins and P-
type families.
1.6.1 C-type lectins
The C-type or Ca2+-dependent lectin family are made up of a number of proteins that
contain a homologous carbohydrate recognition domain (CRD). The CRD for this
family of proteins are made up of 115-130 amino acids where 18 of these are highly
conserved and 14 are identical. A number of different domains are attached to the
CRD, which forms the bulk of the protein. There are three subfamilies of C-type
lectins: endocytic lectins, collectins and selectins.
Endocytic lectins are type II transmembrane proteins that contain a short N-terminal
cytoplasmic domain, a hydrophobic membrane spanning domain and a neck region
where the C-terminal CRD is linked (Lis and Sharon, 1998). The mammalian and rat
hepatic asialoglycoprotein receptors are two examples of endocytic lectins (Lis &
Sharon, 1998). The macrophage mannose binding lectin is another example of an
endocytic lectin. This lectin differs from other endocytic lectins as it is a type I
transmembrane protein ie. the C-terminus is in the cytoplasm. The extracellular matrix
of the mannose binding lectin contains three domains including a cysteine rich domain,
_________________________________________________________________ 15
Chapter 1 Introduction _________________________________________________________________________________
fibronectin repeats (that are similar to type II proteins) and a string of 8 CRD’s close to
the membrane (Taylor et al 1992; Taylor & Drickamer, 1993).
Collectins are soluble proteins that contain a cysteine-rich amino terminus, a region of
collagen-like repeats and a α-helical neck region that contains the CRD’s at the C-
terminus. Examples of these proteins include the serum mannose binding protein
(MBP) A & C (Drickamer et al, 1986, Childs et al, 1990), the pulmonary surfactant
proteins, SP-A and SP-D (Haagsman et al 1987, Childs et al, 1992, Persson et al 1990),
and the CL-43 from bovine serum and bovine conglutinin (Haagsman et al 1987).
Collectins can be distinguished from other lectins by the length of their collagenous
domain. The smaller number of Gly-X-Y repeats have been found to form a bouquet
like structure (MBP-A, MBP-C & SP-A) and larger Gly-X-Y repeats form cruciform
like structures (SP-D) as seen in Figure 1.7 (Weis et. al., 1998).
Figure 1.
(A) The c
A) and (C
________
A
7: A schematic representation of th
ollectin monomer is made up of four reg
) the cruciform (SP-D) of collectins.
______________________________
B
e
io
_
C
collectin proteins.
ns. (B) The bouquet form (MBP-A & SP-
__________________________ 16
Chapter 1 Introduction _________________________________________________________________________________
The mannose binding proteins are made up of a trimer of subunits, formed by the triple
helix of collagenous regions of the protein (Figure 1.8). It is approximately 32 kDa in
size and it is found in the serum for MBP-A (where is circulates as a hexamer of
trimeric units) and in the liver for MBP-C (Rini and Lobsanov, 1999).
A B
Figure 1.8: The structure of the MBP-A taken from the side (A) and the top (B).
The MBP-A is made up of three domains each containing a α-helical region that is followed by
the CRD. Structures were taken from the RSCB PDB database (PDB ID: 1AFA; Kolatkar &
Weis, 1996).
The mannose binding protein functions by directly attaching to pathogens and initiating
the complement cascade, which activates the expression of the MBP-associated
proteases (MASP-1 and MASP-2). The MBP-A recognises the bacteria, Escherichia
coli and Salmonella montevideo and also the fungi in the Candida and Cryptococcus
groups (Weis et. al., 1998; Epstein et. al., 1996).
The surfactant proteins have been found to be the first line of defence against airborne
pathogens that may attach to the fluid lining of the lungs. The SP-A binds to the
Haemophilus influenzae and Streptococcus pneumoniae and the SP-D has been found to
_________________________________________________________________ 17
Chapter 1 Introduction _________________________________________________________________________________
bind to Klebiella pneumoniae and Escherichia coli. Both of these proteins require other
mechanisms to remove the bound bacteria, as they do not have the ability to fix
complement like the MBP’s (Weis et. al., 1998; Epstein et. al., 1996).
Selectins are made up of three proteins that are involved in the migration of
lymphocytes from the lymph node endothelium and the removal of circulating
neutrophils and monocytes to the site of inflammation and infection (Lis & Sharon,
1998). There are three members of the selectin family and they are the L-selectin (found
on leukocytes) P-selectin (platelets) and E-selectin (endothelium) (Weis et. al., 1998;
Lis & Sharon, 1998). These proteins have been discussed earlier in this chapter
(Section 1.2) and will not be discussed in detail here.
1.6.2 I-type lectins
I-type lectins are members of the immunoglobulin superfamily as their extracellular
domain is made up of immunoglobulin-like structures. A number of proteins are
classified as I-type lectins and they are listed in Table 1.2. These proteins are
characterised as type I transmembrane proteins in which the amino terminal in the
extracellular domain is found to be similar to the variable region (V-type domain) of the
immunoglobulin IgG (Gabius, 1997; Lis & Sharon, 1998).
_________________________________________________________________ 18
Chapter 1 Introduction _________________________________________________________________________________
Table 1.2: The members of the I-type lectin family. Name Occurrence Carbohydrate ligand ICAM-1 (CD54) endothelial cells, many activated cell types hyaluronic acid PECAM-1 (CD31) platelets, endothelial cells, myeloid and B lymphoid lineage cells Heparin N-CAM central and peripheral nervous system oligomannoside (and complex ?) glycans Heparin Po glycoprotein peripheral nervous system HNK-1 epitope Myelin-associated glycoprotein peripheral nervous system Neu5Acα2-3Gal Sialoadhesin macrophages in hemopoietic and secondary Neu5Acα2-3Gal lymphoid tissues CD22 mature B cells Neu5Acα2-3Gal CD33 myeloid progenitor cells, monocytes Neu5Acα2-3Gal
1.6.3 Galectins
Galectins are a family of β-galactosidase binding lectins found in mammals and they
consist of 1 or 2 highly conserved carbohydrate recognition domains that are 135 amino
acids long (Hughes, 1999). Galectins are thought to be involved in intra- and
extracellular functions such as modulating cell-cell and cell-matrix interactions (Rini,
1995). These lectins have specificity for galactose, however some galectins are have
been found to be specific for lactose and N-acetyllactosamine. There are four different
structural arrangements including monomers and dimers, and large polypeptides (that
contain 1 or 2 copies of the CRD in association with the repeating linker domains).
Table 1.3 lists the galectins that have been identified.
_________________________________________________________________ 19
Chapter 1 Introduction _________________________________________________________________________________
Table 1.3: The members of the Galectin family.# Name Occurrence Structural Features Galectin -1 (galaptin, L-14) many cell types homodimer; one CRD/subunit (12-16kDa; prototype Galectin-2 lower small intestine; clone from homodimer; one CRD/subunit (14kDa); prototype human hepatoma Galectin-3 (CBP35, Mac-2, IgE- many cell types monomer with one CRD; Pro-,Tyr-, Gly rich repeats binding protein, L-29, L-34 in N-terminal section (29-37kDa); chimera type Galectin-4 colon, small intestine, stomach monomer with 2 partially homologous but distinct CRD oral epithelium, oesophagus connected by a linker region (36kDa); tandom repeat Galectin-5 blood cells monomer with one CRD (17kDa); prototype Galectin-6 small intestine, colon tandom repeat arrangement of two CRDs (33kDa) Galectin-7 keratinocytes one CRD (12.7kDa); prototype Galectin-8 several tissues homologous to galectin-4 & -6 (tandom repeat of two CRDs with unique link peptide: 34kDa) #Taken from Gabius, 1997.
Galectins are divided into three groups based on the differences seen within their
binding specificity in the CRD (the proto-type, the chimera-type and the tandem repeat
groups). The proto-type galectins are small proteins of 15kDa that contain one CRD
and Galectins -1, -2, -5 & -7 & are all members of this group.
The chimera-type galectins are found in mammals only and have an approximate
molecular weight of 30-35 kDa. The C-terminus contains one CRD and a region of
sequence that is rich in proline, tyrosine, glycine and glutamine amino acid residues.
The N-terminus of chimera galectins is not homologous with any other galectins but
there are some regions of similarity with the heteronuclear ribonucleoprotein complex
(hnRNP) to which Galectin-3 is a member of. The last group of lectins in the galectin
family is the tandem repeat group. These lectins Galectin –4, -6, -8, contain two CRD
domains within the single polypeptide chain (Kasai & Hirabayashi, 1996; Hughes,
1999).
_________________________________________________________________ 20
Chapter 1 Introduction _________________________________________________________________________________
The three dimensional structures of galectin 1 & 2 have been found to be very similar to
the legume lectin family however, there is very little sequence homology between the
two families. The structures of galectin-7 and concanavilin A are shown in Figure 1.9
where the positioning of the combination site was different between the lectins but were
located on the same face of the CRD.
F
l
(
c
a
1
P
i
l
t
a
a
b
_
A
igure 1.9: Structural similarities of t
ectins.
A) Galectin-7 monomer unit (PDB ID: 1B
oncanavilin A (PDB ID: 1CN1; Shoham et
ntiparallel β-sheet structures.
.6.4 Pentraxins
entraxins are a group of lectins that ar
e, the subunits are arranged in a pentam
ectins require Ca2+ to bind to their carb
his family and they are the C-reactive
nd the serum amyloid P component (SA
re thought to act in the early stages of
een found to precipitate the pneumoco
________________________________
B
he CRD between the Galectin-7 and legume
KZ; Leonidas et. al., 1998) and (B) demetalised
. al., 1979). Both proteins contain 5 or 6 stranded
e defined by the arrangement of their subunits,
er formation as seen in Figure 1.10 and these
ohydrates. Two proteins have been defined in
protein (CRP) (Kilpatrick & Volanakis 1991)
P) (Steel & Whitehead 1994). These proteins
the defence of the host cell where the CRP has
ccal somatic C-polysaccharide (Gabius, 1997).
________________________________ 21
Chapter 1 Introduction _________________________________________________________________________________
A B
Figure 1.10: The structure of the C-reactive protein (CRP) and the serum amyloid
component (SAP).
(A) The C-reactive protein (PDB ID: 1GNH; Shrive et. al., 1996) and (B) the serum amyloid
component (PDB ID: 1SAC; White et. al., 1994). Note the pentamer arrangement of monomer
units that characterise this group of lectins. These structures were taken from the RSCB PDB
databank.
1.6.5 P-type lectins
There are only two lectins that contain the CRD that defines the P-type lectins. These
lectins are the cation dependent mannose 6-phosphate receptors (CD-MPR) and the
insulin growth factor II/ cation independent mannose-6-phosphate receptor (IGF-II/CI-
MPR). They are type-I transmembrane glycoproteins that contain a single
transmembrane domain, a cytoplasmic C terminus and an extracytoplasmic N-terminus
containing the CRD. The CD-MPR contains a single 159 amino acid domain in the N-
terminus and requires cations to bind with the Man-6-P (with high affinity). The IGF-
II/ CI-MPR is larger than the CD-MPR and it is made up of 15 adjacent but homologous
repeating units with two carbohydrate-binding domains (domain 3 & 9) that will bind
Man-6-P in the absence of cations (Rini and Lobsanov, 1999; Kornfeld, 1992; Lis &
Sharon, 1998).
_________________________________________________________________ 22
Chapter 1 Introduction _________________________________________________________________________________
The structure of the CD-MPR has been determined by X-ray crystallography in the
presence of manganese and Man-6-P. The receptor is a dimer, which has been found to
contain a relatively large interface. Binding studies between the CD-MPR and the Man-
6-P have shown the C2 hydroxyl group, the 6-phosphate group and the manganese ion
to be important for binding. The clustering of residues around the C2 hydroxyl group of
the carbohydrate is conserved in the domains 3 and 9 of the IGF-II/CD-MPR, which
suggests the recognition of the mannose, is important in the functioning of these
proteins.
_________________________________________________________________ 23
Chapter 1 Introduction _________________________________________________________________________________
1.7 Plant lectins
Since their discovery in 1880’s, a large number of plant lectins have been identified and
isolated and are found predominantly in the storage organs of the plant, such as the
seeds (Rüdiger, 1998). In legume lectins, the lectin is localised in the cotyledons, for
the caster bean, the lectin is found in the endosperm and in wheat, the lectin is found
confined to the primary axis of the seed (Van Damme et. al., 1998). Lectins can
constitute up to 10% of the total protein content however, it is more likely to be 0.1-5%
(Sharon & Lis, 1990).
Plant lectins are further divided into subfamilies based on their sequence homology and
their molecular weight and it is common to have a large number of lectins with different
carbohydrate specificity in the same family. Plant lectins are divided into the following
families: legume lectins, monocot mannose binding lectins, chitin binding lectins, type
2 ribosome inhibiting proteins, and the jacalin family.
1.7.1 Legume lectins
A large number of legume lectins have been characterised and the majority of these
lectins are located in the seeds where they make up an astonishing 10% of the total
protein content (Sharon & Lis, 1990). Legume lectins are also found in leaves, stems,
bark and roots but in low amounts. Their carbohydrate specificity varies, however the
majority of these lectins are specific for Man/Glc and Gal/GalNAc. Table 1.4 lists a
number of legume lectins identified, the species that they have been derived from and
their carbohydrate specificity.
_________________________________________________________________ 24
Chapter 1 Introduction _________________________________________________________________________________
Table 1.4: Members of the legume lectin family. Origin Species Carbohydrate specificity
Caesalpinaceae Griffonia simplicifolia Gal/GalNAc, GlcNAc, oligosaccharides Papilionaceae - Abreae Abrus precatorius Gal/GalNAc Papilionaceae - Carageae Caragana arborescens (Pea tree) Man(Glc) Diocleae Canavalia ensiformis (ConA) Man(Glc) Phaseoleae Glycine max (soybean) Gal/GalNAc Phaseoleae Phaseolus lunatus limensis (lima Bean) Gal/GalNAc Phaseoleae Phaseolus vulgaris (kidney bean) oligosaccharides Vicieae Lens culinaris (lentil) Man(Glc) Vicieae Pisum sativum (garden pea) Man(Glc) Vicieae Vicia faba (fava bean) Man(Glc) Vicieae Vicia cracca (common vetch) Man(Glc), Gal/GalNAc Loteae Lotus tetragonalobus (asparagus pea) L-fucose
Legume lectins consist of two or four identical or nearly identical subunits of about 30
kDa, which is composed of 6- or 7-stranded antiparallel β-sheets. Each subunit contains
a single carbohydrate-binding domain that has the same carbohydrate specificity. Most
legume lectins form what is commonly known as the canonical legume monomer as
seen in Figure 1.11. The dimeric form is defined as a large 12 stranded β-sheet that
results from the association of two 6 stranded β sheets as seen in
Figure 1.11B.
_________________________________________________________________ 25
Chapter 1 Introduction _________________________________________________________________________________
___________________
Figure 1.11: The stru
(A) The canonical mono
Banerjee et. al., 1996). S
The formation of the ca
presence of two diva
(PDB ID: 1CN1; Shoham
teracting with the m
lectins also have hydro
that binds to non-pol
pockets often result in
(Sharon & Lis, 1990; V
carbohydrate-binding s
impaired (Sharon & L
in
C
A
__________________
ctures of legume lecti
mer (PDB ID: 2ENR;
tructures were taken from
rbohydrate-binding si
lent cations (Mn
et. al., 1979) and (C) t
etal ions are highly
phobic binding sites
ar compounds such a
a 10-50-fold increas
an Damme et. al., 199
2+
ite is not formed pro
is, 1990). The ami
B
____________________________ 26
ns.
Bouckaert et. al., 1996), (B) concanavalin A
the RCSB PDB database.
te of the legume lectins is dependent on the
he peanut agglutinin tetramer (PDB ID: 2PEL;
id residues that are responsible for
conserved among these lectins. Legume
located near the carbohydrate-binding site
s indoleacetic acid and adenine. These
e in binding of hydrophobic glycosidases
7).
and Ca2+). Without these cations, the
perly and the functioning of the lectin is
no ac
Chapter 1 Introduction _________________________________________________________________________________
1.7.2 Monocot Mannose binding lectins
Monocot mannose binding lectins are a group of proteins that are specific for mannose
nly. They were first discovered in the snowdrop bulbs, and other lectins have been
und in a number of different families including Amaryllidaceae, Alliaceae, Araceae,
rchidaceae and Liliaceae (Van Damme et. al. 1987). The structure of the Snowdrop
ctin complexed with its sugar is shown in Figure 1.12.
igur
mann
from
A
o
fo
O
le
F
(A) E
mann
key re
Mono
have
Hom
the c
of th
prepr
____
e 1.12: The structure of the Snowdro
oside (A) and a close up of the carbohy
the RSCB PDB database (PDB ID: 1MSA; He
rotein into the mature polypeptide. The
ach subunit is made up of primarily β-
osides per subunit bind (shown in yellow). (
sidues involved in forming hydrogen bonds
cot mannose-binding lectins are approxim
been three different types identified (hom
omers contain 2 or 4 identical subunits of
leavage of the signal peptide during trans
e peptide during the post-translational s
op
__________________________________
B
p lectin complexed with methyl α-D-
drate binding site (B).
ster et. al., 1995).
heterodimer was formed using the same
sheet structures where three methyl-α-D-
B) The carbohydrate is shown in yellow and
are shown in red. The structure was taken
ately 12 kDa in size, and to date, there
omer, heterodimer and heterotetramer).
approximately 12 kDa and are formed by
lation and the removal of the C-terminus
tage, resulting in the conversion of the
___________________________ 27
Chapter 1 Introduction _________________________________________________________________________________
methods as the homomer and these polypeptides contain 2 different but very similar
contains two mannose-binding domains. The
eterodimer in the A.sativum lectin (ASA-I) possess two tandomly arrayed highly
homologous domains. The mature heterodimer is formed by the removal of the
glycosylated linker found between the two domains and the non-glycosylated C-
terminus (Van Damme et. al. 1992).
Urticaceae, Solanaceae, Papaveraceae,
uphorbiaceae, Phytolaccaceae and Viscaceae. This domain is a small chitin binding
subunits of approximately 12 kDa that were derived from different preproproteins. The
lectin isolated from Allium ursinum is an example of the heterodimer (Smeets et. al.,
1994).
The second type of monocot mannose binding lectin includes the heterodimer and
heterotetramer forms. They are the result of two different subunits produced from the
same precursor where each subunit
h
1.7.3 Chitin-binding lectins
Chitin binding lectins contain a hevein domain and have been found in a number of
plant families such as Graminceae,
E
protein of 43 residues that was first found in the latex rubber tree Hevea brasiliensis.
An interesting characteristic of this protein is the primary sequence contains a large
number of cysteine and glycine residues.
The Graminceae lectins have been found in Triticum aestivum, T.durum, Secale cereale,
Hordeum valgare and Oryza sativa. All of these lectins have two subunits of 18 kDa
(Peumans & Stinissen, 1983). The barley, rye and rice lectins contain only one isoform
and therefore only one subunit while the wheat germ agglutinin (WGA) is made up of
_________________________________________________________________ 28
Chapter 1 Introduction _________________________________________________________________________________
three isoforms (or three different subunits). Each subunit of the WGA is approximately
17 kDa and contains four homologous subdomains of 43 amino acids that are held
together by 32 disulfide bridges. Each subdomain is arranged in a pseudo four fold
screw-related fashion (Rini, 1995) where the monomers are paired head to tail which
sults in the formation of the dimer (Lis and Sharon, 1998). Proteins within this family
contain a number of different carbohydrate specificities as a result of the variability
between the sequences within each subdomain. For example, WGA is specific for N-
acetylglucosamine and N-acetylneuraminic acid.
ere the lectins specificity is for Neu
Acα(2,6) Gal/GalNAc (Van Damme et. al., 1996; 1997). Lectins that belong to this
family include ricin (Euphorbiaceae), abrus (Abrus precatorius - Fabaceae) and SNA-I
& SNA-V (Sambucus nigra – Sambucaceae) (Peumans and Van Damme, 1998). The
crystal structure of ricin is shown in Figure 1.13.
re
1.7.4 Type II ribosome inactivating protein (RIP)
Type 2 ribosome inactivating protein (RIP) contains two chains (α and β) separated by
a disulfide bond where each chain is made up of 1, 2 or 4 subunits. The α-chain
contains the N-glycosidase activity that results in the cleavage of the rRNA and the β-
chain contains the carbohydrate-binding domain. There is substantial homology
between the sequences for both chains when compared to all type 2 RIPs. The
carbohydrate specificity for type 2 RIP is mainly Gal or GalNAc, however, two cases
have been identified in the Sambucus species wh
5
_________________________________________________________________ 29
Chapter 1 Introduction _________________________________________________________________________________
Figure 1.13: The crystal structure of the ricin A-chain.
Figure was taken from the RSCB PDB database (PDB ID: 1APG; Katzin et. al., 1991).
1.7.5 The Jacalin family
Jacalin is a galactose specific, tetrameric lectin with a molecular weight of 66 kDa,
derived from the Artocarpus integrifolia or the jackfruit. Each subunit is made up of a
heavy (133 residues) and light (20 residues) chain that form up to four-stranded
antiparallel β-sheets (Figure 1.14). These subunits are stabilised by a number of non-
covalent bonds such as hydrogen bonds. This lectin is derived from one precursor that
is modified into the mature form by a series of complex post-translational modifications
(Lis and Sharon, 1998; Van Damme et. al., 1997; Peumans et. al., 1998).
_________________________________________________________________ 30
Chapter 1 Introduction _________________________________________________________________________________ Chapter 1 Introduction _________________________________________________________________________________
___
Fig
(A)
loca
(PD
The
bee
β1,
(4 p
A
___
________________________________ure 1.14: The structure of the Jacalin
The full structure of the jacalin lectin and
ted between the two heavy chains. The str
B ID: 1JAC; Sankaranarayanan et. al., 1996
Jacalin lectin from Artocarpus integri
n found to interact with high specificity
3-GalNAc-α) (Kabir & Daar, 1994). Th
er tetramer) that is located at one end of
________________________________
B
______________________________ 31
lectin.
(B) the light chain is shown in red, which is
ucture was taken from the RSCB PDB database
).
folia is a galactose specific lectin that has
to the α-linked T-antigen disaccharide (Gal-
ere is one galactose-binding site per subunit
the β fold.
______________________________ 31
Chapter 1 Introduction _________________________________________________________________________________
1.8 Roles of lectins in plants
Plant lectins are thought to function either directly within the plant or as a defence
system against pathogen invasion. Within the plant, the lectin may be involved in the
transport of sugars; it can be stored as a source of nitrogen, cell-cell interactions and the
regulation of cell division. Externally, most plant lectins are involved in the defence of
the plant by interacting with surface glycoproteins on the digestive tract of organisms
(Peumans & Van Damme, 1998; Van Damme et. al., 1997).
Several plant lectins have been found to affect the growth and development of insects
when the lectin has been orally ingested. The lectin binds to the carbohydrates on the
surface of the digestive tract resulting in the harmful effects to the insect and these
effects have been studied. It was found that the lectin purified from Phaseolus vulgaris
agglutinin (PHA), binds to the brush border cells of the intestine of insects resulting in
hyperplasia and hypertrophy of the small intestine (Pusztai and Bardocz, 1996). In
addition to this, the ingestion of raw beans of PHA by the insects resulted in nausea,
vomiting and diarrhoea (Peumans & Van Damme, 1998). Therefore, lectins in plants
play a key role in the defence against insect invasion.
_________________________________________________________________ 32
Chapter 1 Introduction _________________________________________________________________________________
1.9 Applications of plant lectins
Plant lectins have gained increasing interest in the last couple of decades, as they are
useful tools in the structural and functional studies of complex carbohydrates such as
glycoproteins. Other applications of plant lectins involve the expression of their genes
in transgenic plants for the production of large quantities of lectin and to develop insect
resistant crops (Van Damme et. al., 1997).
Plant lectins also can be used for the detection, isolation and characterisation of
glycoconjugates from a number of different sources. Specific carbohydrate structures
can be targeted and their isolation can be achieved using these lectins. Lectins can also
be used to induce specific processes in animal or human cells. The activation of
lymphocytes with mitogenic lectins and the expression of cytokines and interleukins are
a few more functions of lectins. At this stage, the concanavilin A and peanut
hemagglutinin lectins have been found to cause these cellular reactions.
Lectins are used as diagnostic tools and some of these uses include the typing of the red
blood cells (ABO system), the tracing of aberrant glycosylation of glycoproteins, and
the histochemical staining of carbohydrates. Potential therapeutic uses of lectins
include immunomodulation and cancer therapy with immunotoxins (Peumans & Van
Damme, 1998).
_________________________________________________________________ 33
Chapter 1 Introduction _________________________________________________________________________________
1.10 Serine Protease Inhibitors
One of the two major proteins that were isolated from the seeds of G.robusta was shown
to be a protease inhibitor. There are four different families of protease inhibitors that
exist in plants and they include the serine protease inhibitor family, the cysteine
protease inhibitors, the metallo-protease inhibitors and the aspartic protease inhibitors.
Protease inhibitors are characterised into these families based on the amino acids
involved within the reactive site of the inhibitor. Table 1.5 lists the families of protease
inhibitors and the protease that they inhibit.
Table 1.5: Families of plant protein protease inhibitors. Family Protease inhibited
Serine protease inhibitors Trypsin and chymotrypsin Soybean trypsin inhibitor (Kunitz) family Bowman-Birk family Barley trypsin inhibitor family Potato inhibitor I family Potato inhibitor II family Squash inhibitor family Ragi I-2/maize trypsin inhibitor family Serpin family Cysteine protease inhibitors Papain, cathepsin B, H, L (phytocystatins) Metallo-protease inhibitors Carboxypeptidase A, B Aspartic protease inhibitors Cathepsin D
Taken from Koiwa, 1997.
Serine protease inhibitors are the largest group of protease inhibitors isolated from
plants. There are eight subgroups within this family that are characterised based on their
sequence homology, the location of their reactive site and their structural characteristics
such as the positioning of disulfide bonds. The majority of these inhibitors have been
_________________________________________________________________ 34
Chapter 1 Introduction _________________________________________________________________________________
extracted from seeds. Two of these protease inhibitors, the Kunitz and Bowman-Birk,
have been extensively characterised (Kunitz 1947; Bowman 1946, 1993; Birk 1961,
1974, 1976, 1985). These inhibitors were first discovered in soybeans and were found
to differ from each other in size, primary sequence, three-dimensional structure and
enzyme inactivation properties (Birk, 1976).
The Kunitz-type (Soybean trypsin inhibitor, SBTI) inhibitor is made up of 181 amino
acids, contains 2 disulfide bridges and inhibits trypsin. The Bowman-Birk protease
inhibitor (BBI) is comprised of 71 amino acids, contains 7 disulfides bridges (located
between C8-C62, C9-C24, C12- C58, C14-C22, C32-C39, C36-C51 & C41-C49) and
inactivates both trypsin and chymotrypsin at two independent sites (Birk, 1976). For
the inhibition of trypsin, the presence of a lysine or arginine in the reactive site is
required. For chymotrypsin, the presence of tyrosine, phenylalanine, leucine or
methionine will inactive this protease (Laskowski & Kato, 1980).
Figure 1.15 shows a number of residues that are conserved between all of the members
of the Bowman Birk family. All of the cysteine residues were conserved throughout the
family and these residues were involved in forming disulfide bridges (Figure 1.15). It
was therefore thought that these residues play an important role in stabilising the
structure of the proteins by forming disulfide bridges.
_________________________________________________________________ 35
Chapter 1 Introduction _________________________________________________________________________________
1 10 20 30 P4 P3
1BBI -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D D E S S K P C C D Q C A C 1PI2 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D E Y S K P C C D L C M C IBB3_DOLAX -- -- -- -- -- -- -- -- D H H H S T D E P S E S S K P C C D E C A C IBB4_DOLAX -- -- -- -- -- -- -- -- -- H E H S S D E S S E S S K P C C D L C T C IBB_PHAAU -- -- -- -- -- -- -- -- -- -- -- -- S H D E P S E S S E P C C D S C D C IBB2_PHAAN -- -- -- -- -- -- S V H H Q D S S D E P S E S S H P C C D L C L C IBB3_SOYBN M C I L S F L K S D Q S S S Y D D D E Y S K P C C D L C M C IBB2_SOYBN -- -- -- M E L N L F K S D H S S S D D E S S D P C C D L C M C
1 2 3 4 31 40 50 60 P2 P1 P1
' P2'
P3'
P4'
P4 P3 P2 P1 P1'
1BBI T K S N P P Q C R C S D M R L N S C H S A C K S C I C A L S PI-II T R S M P P Q C S C E D R I -- N S C H S D C K S C M C T R S IBB3_DOLAX T K S I P P Q C R C T D V R L N S C H S A C S S C V C T F S IBB4_DOLAX T K S I P P Q C G C N D M R L N S C H S A C K S C I C A L S IBB_PHAAU T K S I P P E C H C A N I R L N S C H S A C K S C I C T R S IBB2_PHAAN T K S I P P Q C Q C A D I R L D S C H S A C K S C M C T R S IBB3_SOYBN T R S M P P Q C S C E D I R L N S C H S D C K S C M C T R S IBB2_SOYBN T A S M P P Q C H C A D I R L N S C H S A C D R C A C T R S
4 2 5 6 5 7 61 70 80 90 P2' P3
' P4'
1BBI Y P A Q C F C V D I T D F C Y E P C K P S E D D K E N -- -- -- PI-II Q P G Q C R C L D T N D F C Y K P C K S R D D -- -- -- -- -- -- -- IBB3_DOLAX I P A Q C V C V D M K D F C Y A P C K S S H D D -- -- -- -- -- -- IBB4_DOLAX E P A Q C F C V D T T D F C Y K S C H N N A E K D -- -- -- -- -- IBB_PHAAU M P G K C R C L D T D D F C Y K P C E S M D K D -- -- -- -- -- -- IBB2_PHAAN M P G Q C R C L D T H D F C H K P C K S R D K D -- -- -- -- -- -- IBB3_SOYBN Q P G Q C R C L D T N D F C Y K P C K S R D D -- -- -- -- -- -- -- IBB2_SOYBN M P G Q C R C L D T T D F C Y K P C K S S D E D D D -- -- -- --
7 6 3 1
Figure 1.15: Sequential alignment of the members of the Bowman-Birk inhibitor
family.
Numbers underneath cysteine residues represent the conserved disulfide-bonding pattern for the
family. The scissle bond or P1P1’ site is highlighted in cyan. The serine residues involved in
the inhibition of proteases are highlighted in yellow (for trypsin) and red (for chymotrypsin).
_________________________________________________________________ 36
Chapter 1 Introduction _________________________________________________________________________________
The reactive site of the inhibitor is made up of the scissle bond or P1-P1' bond
(nomenclature derived by Schechter & Berger, 1967) which is highlighted in cyan in
Figure 1.15. In the P1 site, a basic amino acid residue is found and this residue forms a
salt bridge with an acidic amino acid within the S1 pocket of trypsin (Koepke et al,
2000). Trypsin will bind to these residues under normal conditions and hydrolyse the
polypeptide chain at the carboxyl end of this residue. The inhibitor functions by having
the basic amino acid residues on the surface of the molecule, which allows the trypsin to
bind to the residue and blocks the protease’s binding site. This prevents the protease
from binding to other proteins and their function is therefore inhibited and is referred to
as competitive inhibition.
Residues at position P3' and P4' of the trypsin binding region were found to be
conserved within the Bowman-Birk family. These residues are involved in the
formation of a turn between two β-sheet structures. A threonine at the P2 site is highly
conserved throughout all Bowman-Birk inhibitors. A number of substitutional
experiments at this P2 site have shown that threonine is important for the functioning of
the inhibitor (McBride et. al, 1998). A standard inhibitory kinetic assay was established
to determine the effects of residue substitution at the P2 position on their ability to
inhibit the protease (McBride et. al., 1998). It was found that threonine had the lowest
KI value, followed by serine with a 20 fold difference in inhibition when compared to
threonine. It was also found that this site had a preference for small residues as large
aliphatic and aromatic side chain residues were found to have large KI values. The
presence of negatively charged residues such as aspartic acid and glutamic acid also
resulted in poor inhibition. This could be due to the unfavourable electrostatic
interactions within the catalytic triad of the protease (McBride et. al, 1998). The
_________________________________________________________________ 37
Chapter 1 Introduction _________________________________________________________________________________
threonine residue is conserved throughout the Bowman-Birk inhibitor family and has
been shown to be important in the functioning of the inhibitor (McBride et. al, 1998).
The reactive site for chymotrypsin contains a serine in the P1' position and a conserved
leucine residue at P1. Chymotrypsin will mostly bind to hydrophobic residues such as
phenylalanine or leucine in the P1 position. The preference for these residues within
chymotrypsin is due to an uncharged serine residue in the S1 pocket (Koepke et. al,
2000). At position P3', the residue proline is conserved throughout the family. Like the
trypsin-binding region, this position is important in forming the turn between two β-
sheet structures.
Structurally, the Bowman-Birk inhibitor has been described as a “bow-tie” motif as it
contains two independent binding sites. The structure of the inhibitor is shown in
Figure 1.16 (Werner & Wemmer, 1992). Each inhibitory domain is defined to contain a
canonical structural motif which is made up of a β-hairpin or antiparallel β-sheet with a
cis-proline containing type VI turn (Werner & Wemmer, 1992). According to the
structural characterisation of proteins (SCOP) database, the Bowman-Birk inhibitor
superfamily belong to the knottin (or small inhibitors, toxins, lectins) fold family which
is defined as “ disulphide-bound fold; contains beta-hairpin with two adjacent
disulphides” (Murzin et. al, 1995).
_________________________________________________________________ 38
Chapter 1 Introduction _________________________________________________________________________________
___________________________________________
Figure 1.16: The structure of the Bowman-Birk in
The two inhibitory sites are found in the loop regio
structures (in red). Taken from the RSCB PDB prote
Like plant lectins, plant protease inhibitors are also th
plants from insect invasion. Normally, the leaves of
protease inhibitors. It was found that there was an
inhibitors localised within the leaves of plants after
1.10.1 Functional Role of Serine Protease Inhibitors
echanically damaged (Green & Ryan, 1972). In
production of protease inhibitors within the rest of th
nd
993). Feeding trials using protease inhibitors have
Chymotrypsin binding site
m
possibly due to signals being sent from the wou
1
of insects which was thought to be due to the inte
proteases found the gut of the insect (Koiwa, et.
inhibitors play an important role in the defence of the
Trypsin binding site
______________________ 39
hibitor.
n between the antiparallel β sheet
in database (1BBI).
ought to function in the defence of
plants contain very low levels of
increase in the levels of protease
they were attacked by insects
or
addition to this, an increase in the
e plant was also seen and this was
site to the phloem (Pearce et. al.,
shown to slow or stop the growth
raction of the inhibitors with the
al., 1997). Therefore, protease
plant against insect invasion.
Chapter 1 Introduction _________________________________________________________________________________
1.10.2 Applications of Serine Protease inhibitors
Certain protease inhibitors have the ability to prevent or suppress cancer-induced cells
(Kennedy, 1998a). The Bowman-Birk protease inhibitor (BBI) derived from soybeans
was found to be a very effective anticarcinogenic agent. The BBI has been extensively
studied both as a purified BBI and as a concentrated form of BBI (BBIC). The BBIC
was produced by treating the soybean flour with acetone and purified using the Birk
purification procedure described in detail elsewhere (Birk 1976). The chymotrypsin-
press carcinogenesis in three
ifferent species (mice, rats and hamsters)(Kennedy 1998b). The BBIC was also found
to suppress several cancers in different organ and tissue types including the colon, liver,
lung, esophagus, cheek pouch and cell of hematopoietic origin and human trials have
begun using the concentrated form of Bowman-Birk protease inhibitor (Kennedy,
1998b). Specific animal models have been created that shows the suppressive effect of
BBI and or BBIC on carcinogenesis (Kennedy, 1998b).
binding site within the inhibitor was found to be responsible for the anticarcinogenic
effect seen in these experiments (Kennedy 1998b). The dose rates of BBIC showed the
protein to have a high chymotrypsin inhibitory activity (100mg/g) while the trypsin
inhibitory activity was more than half (40mg/g) than that seen for chymotrypsin thus,
suggesting the chymotrypsin inhibitory site is important for function (Kennedy, 1998a).
Both the BBI and BBIC proteins were found to sup
d
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Chapter 1 Introduction _________________________________________________________________________________
1.11 Initial Research
Researchers at the Australian Red Cross Blood Service of Queensland (ARCBS-QLD)
are investigating the isolation of lectins, from a number of different species, that result
in the agglutination of granulocytes. Approximately 700 different species of native
Australian and exotic plants were screened for reactivity towards granulocytes. Of
these, only 36 species were reactive with granulocytes and not with red blood cells
(Minchinton, 1997b). Eight of these species were chosen for further study based on the
availability of the seeds.
A number of preliminary bioassays and characteristics were determined on these 8
different species and these results are shown in Table 1.6. Of these 8 species, the lectins
isolated from Caragana and Arbus are commercially available. The lectin from
Hernandia moerenhoutiana has been isolated by a colleague at the ARCBS- QLD
(Clarke, 1997).
Grevillea robusta was one of the 8 short listed species to be investigated in the future.
The plant was chosen for this study because of: (1) the size of the protein, (2) the
availability of the crude material (ie seeds), (3) the ability to specifically target white
blood cells and not red blood cells and (4) the presence of two major proteins that were
found to fall within the molecular weight range of interest.
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Chapter 1 Introduction _________________________________________________________________________________
Table 1.6: Preliminary biological characteristics of the 8 species of plants. Plant species MW in kDa of SDS Sugar specificity Potential antigen target Potential antigen target PAGE bands (crude - defined by monoclonal defined by human reduced) antibodies antibodies
Grevillea robusta 27, 21, <10 man> malt CD15,CD18, CD65 HNA-1a, HNA-2a NB2* Hernandia moerenhoutianna 28, 26, 24, 23, 16, man=fuc>malt CD15,CD16, CD18, HNA-2a,HNA-4a NB2* 14.5, 14 CD65 Lablab purpureus sweet rongai 78/74,35/31/28.5, gal=galNAc CD11b,CD15, CD65 HNA-1a, NB2*, SL* 19,<10 Erythrina speciosa 84, 76, 28 galNAc>gal >fuc CD15, CD65 HNA-1a Vicia sativa 94, 54, 39, 16 glc=man CD11b,CD15, CD16, HNA-2a CD18, CD24, CD65 Caragana arborescens 100, 25, 13 gal>galNAc incomplete incomplete Lathyrus lirsutus 85, 54, 39 glc>malt incomplete incomplete Arbrus precatorius 58, 54, 39 gal>galNAc CD11b,CD15, CD16, HNA-1a, HNA-1b CD43
* Indicates the neutrophil antigen has not been characterised.
The major proteins identified in the crude extract of G.robusta were found to have a
molecular weight of approximately 7000 Da and therefore would provide an excellent
candidate for structural studies using nuclear magnetic resonance (NMR) spectroscopy.
The preliminary studies revealed the crude extract from G.robusta contained lectin like
properties that were specific towards white blood cells rather than red blood cells. This
species of plant was chosen for further investigation because of at least 1 bioactive
protein with a molecular weight of 7000 Da, which was amenable to NMR studies.
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Chapter 1 Introduction _________________________________________________________________________________
1.12 Aims and Expected Outcomes
There were two main aims to this project; (1) the purification and characterisation of a
proteins from the seeds of a native Australian species that fell within the 10 – 30 kDa
molecular weight range and (2) the determination of the structure of the protein from
that species. The initial focus was to target the lectin identified in the seeds of the
native Australian plant, Grevillea robusta. This focus was altered as the project
progressed due to limited amounts of lectin found within the seeds (less than 5% of the
total protein content) and the discovery of a serine protease inhibitor (major
representative within the crude extract). Therefore, the first aim was to isolate and
characterise the lectin and serine protease inhibitor from the seeds of G.robusta. The
purified lectin would provide an alternative tool in the characterisation of neutrophil
antigens while the serine protease inhibitor may possess anticarcinogenic properties as it
has a high level of similarity with the Bowman-Birk inhibitor.
As the serine protease inhibitor was very similar to the Bowman-Birk inhibitor (both
sequentially and in function), it was decided that the 3D structure of the inhibitor would
be determined. In addition, the 3D structure of the Bowman Birk inhibitor had already
been determined by NMR spectroscopy (Werner & Wemmer, 1992), thus providing an
excellent starting platform for the study of the serine protease inhibitor from G.robusta.
The second aim was to determine the structure of the serine protease inhibitor from the
seeds of G.robusta.
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Chapter 2 Extraction of Proteins from Grevillea robusta _________________________________________________________________________________
Chapter 2 Extraction of Proteins from Grevillea
robusta
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Chapter 2 Extraction of Proteins from Grevillea robusta _________________________________________________________________________________
Chapter 2 Extraction of proteins from Grevillea robusta
2.1 Introduction
Grevillea robusta (or Silky Oak tree) is a member of the Proteaceae family and is
native to Australia where it is localised in the rainforests of Queensland and northern
New South Wales but has also been found in other neighbouring countries. It is
characterised as a tall, straight tree (seen in Figure 2.1) that contains golden
toothbrush-like flowers, seen only during spring. The leaves are pinnate with lobed
segments that resemble a fern and the undersides of the leaves contain silky hairs.
Preliminary experiments have shown the G.robusta seeds to contain a lectin with an
approximate molecular weight of 7000Da with a sugar specificity for
mannose>maltose (Minchinton, 1997b). As stated in the previous chapter, the lectin
from G.robusta has the potential to be used as a tool in characterising neutrophil
antigens and therefore, the purification and characterisation of this protein would be
advantageous.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
Figure 2.1: Grevillea robusta or Silky Oak tree.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
2.2 Ammonium sulfate precipitation of crude proteins.
G.robusta seeds were ground and soaked overnight in phosphate buffered saline
(PBS) pH 7.3 (0.15M NaCl, 11mM sodium phosphate). Ammonium sulfate was
added to the filtered supernatant in two stages resulting in the proteins being
precipitated. SDS (18% separating gel with 3% β-mercaptoethanol) and native
(13% separating gel) PAGE were used to visualise these extracted proteins (Figure
2.2). These precipitated proteins will be referred to as the “crude extract” for the
remainder of the thesis.
Figure 2.2: SDS (A) and Native (B) PAGE of the crude
The gels were stained with Coomassie Brilliant Blue R-250. (
(5% stacking gel) pH 8.8 in the presence of 3% β-mercaptoeth
Lane 1: crude extract from G.robusta. (B) 13% native separati
8.9. Lane 2: crude extract from G.robusta.
_______________________________________________
2
extract from G.robusta.
A) 18% SDS separating gel
anol. M: LMW standards.
ng gel (3% stacking gel) pH
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
SDS PAGE (under reducing conditions, 3% β-mercaptoethanol) was used to
determine the approximate molecular weight of proteins of the crude extract. The
ratio, between the migration of the protein band and the migration of the dye front,
was determined for each of the standards and samples. These values were plotted
against the log of the standard molecular weight and the molecular weight of the
crude extract was calculated using this graph. SDS PAGE of the crude extract
revealed two major bands below 14.4 kDa and a minor band at 21 kDa. The
approximate molecular weight for the major bands was calculated to be 8700 Da &
7000 Da (upper and lower bands respectively).
Native PAGE was used to show the migration of the crude extract under non-
reducing conditions. This form of electrophoresis allows the proteins to migrate
through the gel in their native or “true” state, which is dependent on the overall
charge of the protein, and to a lesser extent, its size. Native PAGE of the crude
extract, seen in Figure 2.2B, showed 6 bands (labelled SO1-SO5 & I). The major
bands were labelled SO1 and SO2. The remaining 3 bands were of equal intensity
and were labelled SO3, SO4, SO5 & I. Due to the increase in the number of
proteins seen using native PAGE compared to SDS PAGE, it was decided to use
native PAGE to identify the proteins during each purification step.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
2.3 N-terminal sequencing of the crude extract
SDS PAGE of the crude extract revealed 2 major species of proteins with a
molecular weight of approximately 7000 Da. Native PAGE of the same sample
showed 5 bands suggesting the crude extract might contain a number of isoforms.
Isoforms are proteins that contain sequences that are homologous with each other
but differ in their primary sequence by a few amino acid residues.
N-terminal sequencing is useful in determining whether a sample contains isoforms
and was used to determine whether isoforms were present in the crude extract of
G.robusta. The five bands seen on native PAGE were transferred onto Sequi-Blot
PVDF membrane for 1 hour at 100V using BioRads Mini Trans-blot equipment.
Coomassie Blue stained bands were cut from the PVDF membrane, washed and sent
to the University of Queensland Biochemistry Department and sequenced for a fee.
Table 2.1 shows the sequencing results of the proteins seen in the crude extract.
Table 2.1: N-terminal sequences of the crude extract from G.robusta.#
SO1 E/ L T N A R W S -- -- -- -- -- -- -- -- -- -- -- -- -- S
SO2 G G E E A D W (C) E D D V V T T S (C) S I P P
SO3 (C) L P N I (C) I S S D L D -- -- -- -- -- -- -- -- --
SO4 L G P/ N/ I/ (C) D/ S S/ D/ L/ -- V/ -- -- -- -- -- -- -- -- S E W I E G ? ?
SO5 S L D (C) I (C) I (C) S D L -- -- -- -- -- -- -- -- -- --
#The one letter code is used to represent the amino acids. Cysteine residues are destroyed
during the sequencing process and are seen as a blank cycle. Therefore the cysteine residues
are placed in brackets.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
The N-terminal sequencing results revealed 5 different sequences (as seen in Table
2.1) which suggested that the additional bands seen on native PAGE were different
proteins rather than isoforms. The preliminary sequencing of the crude extract from
G.robusta was inconclusive due to the lack of resolution between the bands seen on
native PAGE and further purification of this extract will be required to provide good
sequencing results.
2.4 Bioassays
Functional bioassays were developed using granulocytes to identify lectins and they
were the granulocyte agglutination test (GAT), granulocyte immunofluorescence
test (GIFT) and the sugar-blocking GIFT. These bioassays will be discussed in
detail throughout this section. White blood cells were separated from red blood cells
and platelets using EDTA collection tubes and centrifugation. As granulocytes are
heavier than monocytes and lymphocytes (a density greater than 1.077g/ml)
(Olofsson et. al., 1980), granulocytes were further separated from the white blood
cells by using a density gradient and centrifugation. Neutrophils have a very short
life span (they only survive 1-4 days after being released from the bone marrow), the
bioassays were set up immediately after the cells were harvested (Glasser, 1988).
Even though granulocytes are made up of three different cell types, the results seen
in the bioassays are due to the neutrophils response to the addition of the testing
sample and not from the other cells (basophils or eosinophils) (Glasser, 1988).
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
2.4.1 Agglutination
Agglutination distinguishes lectins from other sugar binding macromolecules such
as glycosidases and glycosyltransferases (Lis & Sharon, 1986). The binding of the
lectin to the cells sugar surface receptors results in the activation and migration of
neighbouring cells to the site of infection and it is this migration of neighbouring
cells that causes agglutination. For agglutination to occur, the lectin must form
multiple cross bridges with the cells suggesting a number of sugar binding sites are
required within the lectin (Lis & Sharon, 1986).
Agglutination can be prevented by the size and the number of carbohydrate binding
sites within the lectin, the number and accessibility of receptor sites on the cell
surface, the fluidity of the membrane and the metabolic state of the cells. External
conditions such as temperature, concentration of the cells and the mixing of cells
may also affect agglutination (Lis & Sharon, 1986). All of these factors need to be
taken into consideration when setting up the bioassays.
2.4.2 Granulocyte Agglutination test (GAT)
The granulocyte agglutination test (GAT) is a functional bioassay where the amount
of agglutination is determined by visualisation using a reverse phase microscope.
Granulocytes were diluted to a concentration of 5 x 106 cells/ml in granulocyte
resuspension solution (GRS) to provide a confluent monolayer of cells in the bottom
of a TERASAKI well. The protein sample to be tested was placed (in duplicate)
into each well of the TERASAKI plate under paraffin oil and granulocytes were
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
added to the sample (a ratio of 1:3 (v/v) cells to protein). The plate was incubated at
30°C for 4 hours to allow the agglutination to occur.
The level of agglutination was scored by eye using a reverse phase microscope. No
agglutination (a score of 0) had a confluent layer of cells while for a +1 score, one or
two cells were seen to be interacting with each other, however, the majority of the
cells were still confluent. Lines of cells or small clusters of cells with a large
number of “single” cells gave a score of +2 and as these clusters become larger and
less “single” cells were seen the score was increase to +3. Samples containing large
clusters of cells that look like bunches of grapes with very few “single” cells
resulted in a score of +4. A positive for the GAT bioassay for this work was a score
of 2+ or better.
2.4.3 Granulocyte immunofluorescence Test (GIFT)
The binding of a lectin to the surface glycoproteins on granulocytes can be identified
using flow cytometry. Flow cytometry detects the level of fluorescence of labelled
cells as they flow past a laser beam and can distinguish between red blood cells,
platelets and granulocytes as each cell type has a different physiological make up ie
size and density.
In order to detect the lectin-cell binding, a fluorescent label is attached to the lectin.
To do this, a cofactor (biotin) is required that will bind to the conjugated
fluorochrome (FITC) and also binds to the protein/lectin. Biotin is a common
reagent used for the immunolabeling of antigens in histochemical, blotting and
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
multiwell assays. It irreversibly binds to the lectin/protein through an amide group
(such as lysine) as seen in Figure 2.3.
Figure 2.3: The biotinylation of proteins.
The biotin binds irreversibly to the primary amine groups of lysine in a protein.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
Biotin has a high affinity for avidin (and its derivatives). Unfortunately this affinity
can be too strong and non-specific binding to the membrane can occur. To get
around this problem a derivative of avidin, such as streptavidin, is used to reduce the
amount of non-specific binding. Avidin and streptavidin are commercially available
unconjugated and conjugated to enzymes, fluorochromes and colloidal gold for the
use in a number of immunoassays.
Avidin is a homotetramic protein that contains four identical subunits and each of
these subunits has the capacity to bind one molecule of biotin and therefore, one
avidin molecule will bind 4 biotin molecules. Streptavidin is derived from
Streptomyces avidinii and can also bind up to 4 biotin molecules. It is this form of
avidin that is used in this bioassay.
The flow cytometer is set up to detect granulocytes based on its size and density. A
schematic diagram outlining the GIFT bioassay is shown in Figure 2.4. The level of
fluorescence detected by the flow cytometer is the mean channel fluorescence
(MCF) and is calculated from the peaks seen in the histogram (Figure 2.4D). The
larger the MCF value, the stronger the lectin binds to the granulocyte.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
Figure 2.4: The schematic representation of the GIFT bioassay.
(A) The biotin labelled lectin protein is added to the granulocyte. (B) The lectin binds to the
specific sugar receptor on the surface of the granulocyte. (C) The streptavidin-FITC label is
added which binds to the biotin on the lectin protein. (D) The flow cytometer detects the
level of fluorescence and places it in a histogram.
Each batch of granulocytes harvested will have a different MCF value for the blank
control value on the flow cytometer. The Relative MCF (Rel. MCF) allows the
results to be quantitated between experiments. By taking the MCF value for the
sample and dividing it against the MCF value for the blank, the relative MCF of the
sample is determined. Any Rel. MCF value that is above 4 is regarded as a positive
for binding in this bioassay.
Lectin
Biotin
Streptavidin-FITC
Granulocyte Sugars
A B C
Log Fluorescence
Number of cells counted
D
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
The sample to be tested in the GIFT bioassay is usually diluted to various
concentrations to identify the point of saturation with the granulocytes. This
information provides the optimal working conditions for the sample in which will be
used in other bioassays. The concentration on the sample is also important for
getting good and useful results in the GIFT bioassay. If the protein sample is too
concentrated, the cells will aggregate or agglutinate rendering the experiment
useless, resulting in variation throughout the experiment. It must be noted that the
principle behind the GIFT assay is to study the binding of the lectin to the surface of
the cells. If the cells have agglutinated, there is no way of determining this binding
and future inhibitory experiments used to determine specificity will also be invalid.
2.4.4 Sugar blocking granulocyte immunofluorescence test
Sugar-blocking studies were undertaken to determine the sugar specificity of the
crude extract. The sugar blocking GIFT bioassay provides information on which
sugar the lectin is targeting on the surface of the granulocyte and provides
information for the purification of the lectin proteins from the crude extract.
Lectins are sugar binding proteins that contain at least two binding sites (in the GAT
bioassay, two or more binding sites are required for agglutination to occur). An
inhibitory effect can been detected by the flow cytometer if the sugar binding sites
within the lectin are blocked with their specific sugar before the sample is added to
the cell suspension. Under these conditions, the lectin would not be able to bind to
the surface of the granulocyte and therefore a decrease in the MCF value would be
seen.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
As the aim of this bioassay is to block all sites within the lectin, thus preventing the
lectin from binding to the surface of the granulocytes, it is important to ensure that
the lectin concentration is not too high. If too many binding sites are available even
the highest concentration of sugar will not be sufficient to block all of the sites.
Therefore, the GIFT bioassay should be completed prior to the sugar blocking
bioassay where the sample is diluted to determine the point of saturation of the lectin
on the granulocytes. Once this point is known, the dilution determined is used for
the sugar blocking GIFT bioassay.
The biotinylated proteins were added to eight different sugars at three different
concentrations (0.1M, 0.25M, 0.5M). The ratio of protein to sugar was 1:4 (relative
v/v) and the samples were incubated for 30 minutes, which ensured all of the
binding sites within the protein were filled with the specific sugar. The sugars used
were a combination of monosaccharides (fucose, galactose, N-acetylgalactosamine,
glucose, N-acetylglucosamine & mannose) and disaccharides (lactose & maltose).
Figure 2.5 shows the schematic flow chart of this bioassay.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
A
Figure 2.5: Schematic representation of the sugar blocking GIFT bioassay.
(A) The lectin binds to the granulocyte surface without the addition of sugar. (B) Sugar is
added which blocks the binding site of the lectin and prevents it from binding to the surface
of the granulocyte. (C) The migration of the peak detected by the flow cytometer is an
indication of sugar specificity for the lectin.
The sugar specific for the lectin will occupy its binding site and prevent it from
binding to the surface of the granulocyte. The flow cytometer is set up to detect the
granulocyte and the level of fluorescence. The decrease in MCF indicates the lectin-
binding site being occupied by its specific sugar as seen in Figure 2.5B. The peak in
the histogram will move towards the left indicating the successful blocking of the
lectin-binding site (Figure 2.5C).
Biotin - Lectin
BStreptavidin-FITC
No binding
Sugars
Granulocyte
B
C
Number of cells counted
A
Log Fluorescence
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
2.4.5 Bioassay results for the crude extract of G.robusta
The crude extract from the ground seeds of G.robusta were found to be reactive with
granulocytes in both the GAT and GIFT bioassays as seen in Table 2.2. The extract
was reactive in the GAT to a dilution of 1/8 and the GIFT bioassay revealed strong
binding to the granulocyte surface receptors, as seen by the increase in the MCF
value.
Table 2.2: The GAT and GIFT results of the crude extract from G.robusta.#
GAT GIFT Dilution Score Rel. MCF Neat 4+ -- 1/2 3/4+ 23.61 1/4 3/4+ 23.16 1/8 3+ --
# The GAT bioassay is scored from 0-4+ and the Rel. MCF value indicated strong binding in the GIFT bioassay.
The sugar specificity of the crude extract was determined using the sugar blocking
GIFT assay. The crude extract was diluted ¼ with Bornes BSA buffer and 8
different sugars were added (using different concentrations) to the crude extract
prior to mixing with the neutrophils. The results are shown in Table 2.3.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
Table 2.3: Sugar-blocking GIFT results of the crude extract from G.robusta.#
Sugar Rel. MCF value with Rel. MCF value with 0.25M sugar 0.5M sugar
fucose 25.34 23.62 galactose 20.96 19.14
N-acetylgalactosamine 26.55 24.83 glucose 27.16 28.71
N-acetylglucosamine 25.17 25.26 lactose 34.22 22.84 maltose 26.03 25.43 mannose 16.98 10.09
#Only two different sugar concentrations are shown. All sugars are D-sugars unless
otherwise indicated. The sugar specific for the lectin in G.robusta is shown in bold.
The Relative MCF values are determined by dividing the MCF value obtained by
the sample with the MCF value obtained for the blank. In this case, the blank
sample would be the diluted crude extract incubated with no sugar. The crude
extract incubated with mannose showed the largest decrease in Rel. MCF value (ie.
a shift to the left was seen in the histogram) when compared to the other sugars
(Refer to Table 2.3). These results were different from the preliminary findings
listed in Table 1.6, where the crude extract was shown to be specific for man>malt.
However, in this work, it is evident that the crude extract was not specific for
maltose, as the Rel. MCF values were high (refer to Table 2.3). Therefore in this
work, the crude extract from Grevillea robusta was found to be specific for only
mannose.
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Chapter 2 Extraction of Proteins from Grevillea robusta _____________________________________________________________________________________
2.5 Conclusion
The crude extract from G.robusta contained at least 5 different proteins. The crude
extract was reactive in both GAT and GIFT bioassays and the sugar-binding GIFT
assay revealed the proteins to be specific for mannose only. This result was
different from the preliminary findings (Refer to section 1.11; Minchinton, 1997b)
where it was suggested that the crude extract from G.robusta were specific for
mannose> maltose. Native PAGE was established as the method of choice for the
visualisation for future purification stages.
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________
Chapter 3 Purification & characterisation of a lectin
isolated from the seeds of G.robusta
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________
Chapter 3 Purification and characterisation of a lectin isolated from
the seeds of Grevillea robusta.
3.1 Introduction
Lectins are ubiquitous in nature and can be relatively easy to isolate from the natural
source. The functional role and biochemical characteristics of these lectins are
discussed in Chapter 1 (sections 1.5 to 1.9) and will not be discussed here. The most
successful method used for the purification of lectins from crude plant extracts is
affinity chromatography (Padma et al, 1999, Moreira et al, 1997, Machuka et al, 1999)
Therefore, it was the method of choice to isolate the protein from G.robusta. Affinity
chromatography allows proteins to be separated on the basis of their specificity for a
subunit and many lectins have been isolated using affinity chromatography. A large
number of affinity chromatography resins with different sugar specificities are
commercially available.
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
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3.2 Purification of the lectin from G.robusta
The lectin in G.robusta has been shown to be specific for mannose>maltose
(Minchinton, 1997). A mannose-agarose column (10ml) was used and equilibrated in
PBS pH 7.3. An aliquot (1ml) of crude extract was injected onto the column and the
column was washed several times to ensure all of the non-binding material had been
removed from the column. The elution buffer (0.5M mannose in PBS pH 7.3) was
added. It was found that the lectin in the crude extract did not bind to the column as no
peak was seen after the addition of the elution buffer.
The lack of binding to the column could result from the lectin not containing a
functional or intact binding site, due the metals ions being lost during the extraction
stage. Another possibility involves the sugar specificity of the lectin. Some lectins
require more complex sugars, such as oligosaccharides or polysaccharides that are
attached to the resin before they will bind.
Other lectins (such as those in the legume family) require metal ions to form their
binding site (Sharon & Lis, 1990; Rini, 1995). This could be the case with the lectin
from G.robusta. During the extraction process, metal ions could have been removed by
the addition of an acid or Na-EDTA. If these metal ions have been removed, the lectin-
binding site will no longer be formed thus rendering the protein inactive and unable to
bind to sugars. Sharon and Lis (1990) showed that these metal ions could be re-
introduced to the lectin in order to re-fold the binding site. However, the metal ions
must be added in a specific order to ensure the correct refolding of the binding site.
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________
To test this hypothesis, metal ions were added to the crude extract (50µl of 0.5M metal
ion solutions/ 100µl of protein solution), in the order of Mn2+ and then Ca2+, and
allowed to incubate for 30 minutes (Sharon & Lis; 1990). This mixture was injected
onto the mannose-agarose column to determine whether the binding site of the lectin
was inactive or the specificity of the lectin required more complex structures for its
binding. The column was run using the same conditions as before. The addition of
metal ions to the crude extract did not improve the binding of the lectin to the resin
(results not shown).
Therefore it was proposed that the lectin from G.robusta did not bind to the mannose-
agarose resin due to its specificity for more complex sugar molecules rather than
monosaccharides. An oligosaccharide-agarose affinity chromatography resin (mannan-
agarose resin) was purchased and used in the attempt to isolate this lectin. This resin,
which is made up of many mannose units bound together, was equilibrated with PBS
pH 7.3. The crude extract from G.robusta was injected onto this column and washed
with PBS pH 7.3 until the UV trace returned to the baseline. This peak represented the
unbound material (P1). The bound protein (P2) was eluted using 0.5M mannose in PBS
pH 7.3 as seen in Figure 3.1. Previous experiments using the crude extract from
G.robusta have shown the extract to contain a lectin that was specific for mannose and
therefore it was used to elute the lectin from the column.
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
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Figure 3.1: Affinity chromatography of the crude extract from G.robusta.
A mannan-agarose column was equilibrated in PBS pH 7.3 and 0.5M mannose in PBS pH 7.3
was used to elute the bound proteins. Two peaks were seen and labelled P1 & P2.
When comparing the sizes of the two peaks seen in Figure 3.1, it could be safely stated
that the lectin protein isolated from G.robusta could constitute approximately less than
5% of the total protein content. These results are consistent with a number of lectins
that have been isolated from seed material (Rüdiger, 1998; Moreira, R, et. al., 1998).
Native PAGE was used to identify the proteins within the eluted fractions in the attempt
to identify the fraction containing the lectin (Figure 3.2). P1 contained all of the bands
seen in the crude extract (SO1-SO5) while P2 contained the SO1 band with a few faint
bands (SO3 & SO5). Therefore, the lectin was identified as the SO1 band seen on
native PAGE.
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________
Figure 3.2: Native PAGE of the eluted fractions.
Crude extract was prepared in PBS pH 7.3. Lane 1: P1. Lane 2: bound protein, P2.
3.3 Bioassays
d there was evidence of some binding in the GIFT assay. The absence of
that all of the lectin was removed from the crude extract.
Review g the bioas o ation was found that the amount
of mate i uir r the lutinatio granulocytes was much less for
the eluted lectin when comp ude extract. As the amount of lectin found
in the c ude extract is less than 5% of e total p functioning ability of the
A 13% separating gel (with a 3% stacking gel) was made and stained with Coomassie Blue. C:
The two peaks from the mannan-agarose column were diluted to 1:20 and tested in the
GAT and GIFT bioassays to determine which fraction contained the lectin. The GAT
bioassay showed the lectin to be present in P2 and this was confirmed with the GIFT
bioassay. The results are shown in Table 3.1.
The protein concentration was determined using the Bradford protein estimation
protocol (refer to section 10.3). P2 was found to agglutinate neutrophils in the GAT
bioassay an
agglutination and low Rel. MCF values were seen for P1 which indicated the lectin was
not present in this sample and
in say and protein c ncentr results it
rial or prote n req ed fo agg n of
ared with the cr
r th rotein, the
_________________________________________________________________ 67
Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________ lectin would be im sev nteracting proteins within this
extract. Therefore, it was not surprising to find the purified lectin to result in a stronger
reaction with the gra yte en co ared to the crude extract.
able 3.1: GAT and GIFT bioassay results of the proteins eluted from the
mannan-agarose column. #
paired as there would be eral i
nuloc s wh mp
T
Dilution [Protein] GAT GIFT (mg/ml) Score Rel. MCF Crude extract Neat 2.40 1+ 5.23 1:2 1.20 3+ 4.35 1:5 0.48 1+ 1.95 P1 Neat 1.48 0 2.65 1:2 0.74 0 1.83 1:5 0.30 0 1.25 1:10 0.15 0 1.02 1:20 0.07 0 1.02 P2 Neat 0.65 2+ 12.33 1:2 0.33 2+ 9.45 1:5 0.13 1+ 6.01 1:10 0.07 0 1.47 1:20 0.03 0 1.16
#The GAT score is graded from 0 – 4+ and the Rel.MCF values above 4 indicate a positive result
in the GIFT bioassay.
The sugar specificity of the lectin was confirmed by using the sugar blocking GIFT
bioassay. The fraction containing the lectin (P2) (final concentration of 0.33 mg/ml)
was mixed with 8 different sugars (in a 1:4 v/v ratio of protein to sugar) using two
different sugar concentrations (0.25 M & 0.5 M). Both monosaccharides and
disaccharides were chosen for the study to determine whether the protein had a
preference for simple or complex sugars. The results of the sugar blocking GIFT assay
are shown in Table 3.2.
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________ Table 3.2: Confirmation of the sugar specificity of the lectin.†
Rel. MCF Rel. MCF 0.25M sugar 0.5M sugar lectin & buffer 28.74 -- fucose 25.83 24.47 galactose 29.42 * N-acetylgalactosamine 28.83 21.65 glucose 29.61 24.47 N-acetylglucosamine 29.03 20.78 lactose 28.83 22.62 maltose 28.74 25.73 mannose 21.75 16.70
† The bold value indicated the sugar that the lectin was specific for. A decrease in the Rel.MCF
value indicates blocking of the carbohydrate-binding site within the lectin. * The sample was
inconclusive.
To determine the sugar specificity of the lectin, a decrease in the Rel. MCF value would
be detected using a flow cytometer. This was seen in the sample incubated with 0.25 M
and 0.5 M mannose. These results confirmed the previous findings when the crude
extract was tested. Also there were no other sugars that blocked the binding site of the
lectin and it was therefore concluded that the lectin isolated from G.robusta was specific
for mannose only and not mannose/glucose or mannose/maltose as previously suggested
(Minchinton, 1997b).
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________
3.4 N-terminal sequencing of the lectin.
The purified lectin was N-terminally sequenced to determine whether it was similar to
the previously determined GR1 protein. The sample was sequenced three times and
these results were compared with the GR1 protein sequence as seen in Figure 3.3.
Lectin Seq1 -- ?L/ N/ E/ A/ D/ ? P/ E D D V V T ? ? P I P -- ?V Q D R E ? W Lectin Seq2 G? G? N E A/ D/ W C? E D D V V T R -- -- -- -- R E Lectin Seq3 ?G/ G? D/ E A D W C? E D D V V T R/ -- -- -- -- ?A E T Lectin Seq4 ? ? P E A D ? S E D D V ? T/ ? A -- -- -- G GR1 protein G G E E A D W C E D C V C T R S I P P
Figure 3.3: N-terminal sequencing results of the lectin.
The results were compared with the cDNA-determined sequence of GR1 protein. The lectin
was sequenced 3 times (Lectin Seq1-3) and lectin Seq4 represents the minor band (SO3) seen
on native PAGE. The yellow highlights indicated similarities between the GR1 protein and the
lectin.
The regions of similarity between the N-terminal sequence and the GR1 protein are
highlighted in Figure 3.3. Three separate sequencing experiments were carried out
which corresponds to the three sequences in the figure labelled as lectin Seq 1-3. The
sequence labelled Lectin Seq4 corresponds to the contaminating band seen on native
PAGE (SO3) as seen in Figure 3.2.
There were similarities between the N-terminally derived sequences for the lectin and
the GR1 protein. The conserved residues (indicated in yellow within the figure)
demonstrated a 50% homology throughout all of the N-terminal sequences. While there
is insufficient evidence to state the lectin and the GR1 protein are the same protein;
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________ there is sufficient data to suggest there are significant regions of similarity between the
two proteins. The level of similarity between the two proteins will not be determined
until the full amino acid sequence of the lectin has been defined. Unfortunately, the
same strategy used to determine the sequence of the GR1 protein cannot be replicated as
the N-terminal sequences for the GR1 protein and the lectin are too similar. Another
method will have to be applied to determine this sequence, such as sequencing using
digestive proteases and liquid chromatography-mass spectroscopy (LC-MS).
The lectin Seq4 was found to contain 50% sequence identity with the lectin Seq 1-3
proteins and 44% sequence identity with the known GR1 protein. Therefore the band
corresponding to SO3 could be a product of carboxy-terminal cleaving of the GR1
protein as the N-terminal sequencing results showed substantial homology with the
lectin Seq1-3 N-terminal sequences.
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Chapter 3 Purification & Characterisation of a lectin isolated from the seeds of G.robusta
_____________________________________________________________________________________
3.5 Conclusion
The lectin was purified from the seeds of G.robusta by using affinity chromatography.
Previous attempts to use this method were unsuccessful due to the lectins’ lack of
specificity for monosaccharides. A multi-branched mannose matrix was used to bind
the lectin from the crude extract and the lectin was eluted using mannose. Native PAGE
revealed the lectin to correspond to the band SO1. The amount of lectin protein isolated
from the crude extract of G.robusta was estimated to be less than 5% and this was
determined by the results from the purification and by native PAGE. These results were
consistent with other lectins isolated from seeds. Bioassays identified the fraction
containing the lectin and also aided in the quantitation of this lectin. The specificity of
the eluted lectin was confirmed with the sugar-blocking GIFT bioassay. This bioassay
also confirmed the lectin from G.robusta to belong to the monocot mannose binding
family. N-terminal sequencing of the lectin revealed a 50% sequence identity with the
previously determined GR1 protein. The full amino acid sequence of the lectin will
have to be determined using alternative methods due to the similarity of the N-terminal
sequences and the GR1 protein. The 3D structure of the lectin could not be determined
because the total lectin concentration within the seeds was very low. In order to
increase the protein concentration required for NMR spectroscopy, a number of
different experiments, such as recombinant protein expression, would need to be
completed. The lectin isolated from the seeds of Grevillea robusta was named the GR2
protein.
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Chapter 4 Purification of the GR1.HPLC protein
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Chapter 4 Purification of the GR1.HPLC protein
4.1 Introduction
Affinity, ion exchange (IEX), gel filtration (GF), hydrophobic interaction
chromatography (HIC), hydroxyapatite (CHT II), and reverse phase high-pressure liquid
chromatography (RP-HPLC) were used in the purification of the proteins from the crude
extract of G.robusta. All of the chromatography methods were completed using the
Biologic HR chromatography system from Bio-Rad Laboratories with the exception of
the RP-HPLC chromatography, where the Waters HPLC system was used. The
Biologic HR system is a medium pressure liquid chromatography system that contains
an in-line UV detector (wavelengths 254 & 280nm) and in-line conductivity cell. Both
of these detectors were useful in monitoring and identifying eluting proteins in each
chromatography method.
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
4.1 Initial Purification of the Proteins from the Crude Extract of G.robusta
4.1.1 Gel Filtration (GF) Chromatography
Gel filtration chromatography separates proteins on the basis of their size. The resin is
made up of tiny beads that contain pores that allow proteins/compounds to enter and
exit. A number of different pore sizes are available to accommodate any size protein
molecule or macromolecule.
SDS PAGE of the crude extract from G.robusta revealed 2 major bands of
approximately 8700 Da & 7000 Da and several other bands of approximately 14 kDa,
28kDa (Figure 3.2A). A sephacryl S-200 HR resin (Pharmacia) (with a pore size of
5000 – 250000 Da) was used to separate the higher molecular weight proteins from the
lower molecular weight proteins. A S-200 column (45ml) was made and equilibrated in
PBS pH 7.3 at a flow rate of 1.5ml/min. An aliquot (1ml) of crude extract was injected
onto the column. Three peaks were detected (by absorbance at 280nm) as seen in
Figure 4.1. SDS PAGE and native PAGE was used to identify these eluted proteins
(Figure 4.2).
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Figure 4.1: Eluted proteins from G.robusta using gel filtration chromatography.
The column was run in PBS pH 7.3 at a flow rate of 1.5ml/min. UV (at a wavelength of
280nm) was used to monitor the elution profile. Three peaks were identified and labelled Pk1-
Pk3.
Two major bands were seen on SDS PAGE for Pk2. This pattern of bands was seen for
the crude extract. This fraction was run on native PAGE (under non-denaturing
conditions) that resulted in the 5 bands seen previously and these bands are labelled
SO1-5 (Figure 4.2B). The GAT bioassay identified the lectin protein to be in Pk2
(Figure 3.1: GAT results are not shown).
Gel filtration chromatography separated the higher molecular weight proteins from the
lower molecular weight proteins (8700Da & 7000Da) and provided information on the
location of the lectin protein. However, the separation of these proteins (two major
bands on SDS PAGE and five bands on native PAGE) required other chromatography
methods.
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Figure 4.2: SDS PAGE (A) and Native PAGE (B) of eluted GF.S200.Pk2 from gel
An 18% SDS separating (5% stacking) gel at pH 8.8 in the presence of 3% β-mercaptoethanol.
M: Low molecular weight standar
filtration chromatography.
ds. Lane 1: crude extract from G.robusta. Lane 2: Pk2 from
e S-200 column. (B) A 13% native separating (3% stacking) gel at pH 8.9 under non-reducing
conditions. Bands are labelled SO1- SO5. Lane 3: crude extract. Lane 4: Pk2 from the S-200
ine groups (-N+(CH3)3) on the resin allows the binding of
th
column.
4.1.2 Ion Exchange (IEX) Chromatography
Ion exchange chromatography separates proteins on the basis of their net charge and is
available in both positively (anion) and negatively (cation) charged functional groups.
These functional groups are covalently linked to a solid support (matrix) to provide the
anion and cation exchanger.
A strong anion exchange resin (Q-sepharose FF, Amersham Pharmacia Biotech) was
used where the quaternary am
negatively charged proteins. These bound proteins can be removed from the resin by
the addition of a competing ion such as Cl- from NaCl. A Q-sepharose FF column
(10ml) was made and equilibrated in 20 mM Tris at pH 8.0. One ml of crude extract
_________________________________________________________________ 77
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
from G.robusta was added and a salt gradient (0-15%, 15-40%, 40-100% 2 M NaCl in
20 mM Tris pH 8.0) was used to elute bound proteins. Each step in the gradient had an
isocratic flow step between each stage, which was equivalent to one column volume.
This allowed the weaker binding proteins to elute during each step without being
contaminated with proteins that eluted later. The elution profile is shown in Figure 4.3.
Eluting peaks were detected by A280 nm. Three peaks were detected and the non-binding
proteins or positively charged proteins were located in Pk1 and Pk2 & Pk3 eluted with
approximately 280 mM and 600 mM NaCl respectively. The irregular shape of Pk2
suggested that this fraction contained multiple proteins and therefore, this method must
be further modified to improve resolution of these bound proteins. This cross
contamination of Pk2 & Pk3 was due to insufficient resolution during the 15-40%
gradient step and this was improved by reducing the concentration of the elution buffer
and eluting the proteins over a shorter gradient.
Figure 4.3: Anion exchange chromatography of eluted proteins from G.robusta.
A step gradient (0-15%, 15-40%, 40-100%) 2 M NaCl in 20 mM Tris pH 8.0 at a flow rate of
1ml/min was used to elute bound proteins. Three peaks were identified and labelled (Pk1-3).
_________________________________________________________________ 78
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
The salt concentration of the elution buffer was decreased to 1M NaCl and more steps
were added (0-14%, 14-35%, 35-55%, 55-100% 1 M NaCl in Tris pH 8.0) to allow
better separation of the proteins in Pk2. The pH, sample volume injected, flow rate
(1ml/min) and buffer composition remained the same. The proteins eluted under these
conditions are shown in Figure 4.4 revealing five peaks (Pk1- Pk5).
Figure 4.4: The elution profile of the crude extract after modifications to the
Proteins were eluted using the gradient 0-14%, 14-35%, 35-55%, 55-100% 1 M NaCl in 20 mM
Tris pH 8.0. T
elution buffer and gradient.
he flow rate was 1 ml/ min. Five peaks were detected and labelled Pk1-Pk5.
SDS PAGE was used to visualise the eluted proteins. These chromatography steps were
completed before the native PAGE results were established as the tool for identifying
the eluted proteins. Pk2 eluted with 140 mM NaCl and contained the two major bands
characteristic of the crude extract from G.robusta ie the proteins of interest. Pk3 also
contained these bands but they were very faint (results not shown) and this fraction
eluted with 300 mM NaCl. Therefore, the proteins of interest were located in Pk2 that
eluted within the range of 140-300 mM NaCl. This method needs to be improved in
order to separate these proteins from each other and this could be done by reducing the
_________________________________________________________________ 79
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
concentration of the elution buffer (from 1 M to 0.5 M) or by increasing the length of
the gradient step (14-35% B).
Using the same column, the eluting salt concentration was further decreased (to 0.5 M
NaCl), the step gradient was altered (0-12%, 12-23%, 23-35%, 35-100%) and the flow
rate was increased to 1.5 ml/min. The increase in the flow rate was due to the increase
in the time taken to run the experiment due to the additional step in the gradient. One ml
of crude extract was injected onto the column (as before) and the results are shown in
Figure 4.5.
Figure 4.5: Modifications to the salt concentration, flow rate and gradient
increase in the flow rate (to 1.5 ml/min) and the addition of a step in the gradient (0-12%, 12-
conditions.
A decrease in the salt concentration of the eluting buffer (0.5 M NaCl in 20 mM Tris pH 8.0), an
23%, 23-35%, 35-100%). Five peaks were detected and labelled Pk1-5.
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
The chromatograph revealed 5 peaks labelled Pk1-5 (Figure 4.5) and native PAGE was
eins being positively charged (ie unable to
ove through the gel).
used to visualise these eluted peaks (Figure 4.6). Pk2, 3 & 4 eluted with 115, 115 &
160 mM NaCl and native PAGE revealed an additional band (labelled I as seen in figure
3.5) within Pk2 along with the band SO3. Pk3 contained SO1/2 SO4/5 and Pk4
contained GR1. The non-binding protein (Pk1) did not reveal any bands on native
PAGE, which could have been due to the prot
m
Figure 4.6: Native PAGE of the eluted peaks from IEX chromatography.
A 13% discontinuous separating gel (with a 3 % stacking gel) was made under non-reducing
conditions. The gel was stained with Coomassie Blue. C: crude extract. Lanes 1-5: Pks1-5.
Two small-scale experiments were completed to determine whether all of the proteins in
G.robusta could be further separated using ion exchange chromatography. The gradient
was further modified and the composition of the buffer was changed. Native PAGE
was used to identify the fractions eluted during each experiment. The conditions and
results of each experiment are listed below.
odified from 0-12% to 0-17%, which would improve the separation
A Q-sepharose FF column (10ml) was made and equilibrated in 20 mM Tris pH 8.0.
The gradient was m
_________________________________________________________________ 81
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
of the proteins between Pk2 and Pk3 as seen in Figure 4.5. The buffer composition and
elution buffer (0.5 M NaCl in 20 mM Tris pH 8.0), the flow rate (1.5 ml/ min) and the
volume of sample injected (1ml) remained the same. Five peaks were detected and the
results are shown in Figure 4.7.
Figure 4.7: Adjustment of the gradient conditions using a 10ml Q -sepharose FF
he flow rate was 1.5 ml/min and gradients used were 0-17%, 17-23%, 23-35%, 35-100% 0.5
M NaCl in 20 mM Tris pH 8.0. Five peaks were detected and labelled Pk1-5.
he increase in the gradient resulted in the separation of Pk3 from Pk4 (eluted with 100
n of the proteins from G.robusta when compared to the previous
onditions.
column.
T
T
mM & 115 mM respectively); however, the gradient was not sufficient to separate Pk2
from Pk3 (eluted with 85 mM & 100 mM respectively). Pk2 contained a split peak and
two fractions were taken and identified (labelled Pk2A & Pk2B). Native PAGE showed
Pk2A to contain the proteins SO3 and I where Pk2B contained both of these proteins
plus SO1. Pk3 contained SO1/2 and SO4 and Pk4 contained GR1. This method did not
improve the separatio
c
_________________________________________________________________ 82
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Reviewing all chromatography methods and conditions, it was concluded that the
proteins from G.robusta were of similar charge. Purification of these proteins in one
step was not possible and each fraction would have to be further processed. It was
noted that specific proteins detected by native PAGE would elute together. The
isolation of these groups of proteins was the focus of the initial purification step of the
crude extract from G.robusta.
4.2 Large scale preparation of the crude extract from G.robusta
The best conditions for the isolation of three groups of proteins were identified and
applied to a larger scale purification protocol. Tris (20 mM, pH 8.0) was chosen as the
buffer for it was found that phosphate buffers could chelate metal ions from proteins
aintain
biological activity.
The large scale Q-sepharose FF method used the gradient 0-12%, 12-23%, 23-35%, 35-
100% with 0.5 M NaCl in 20 mM Tris pH 8.0 as the eluting buffer as it provided the
best separation of the proteins. A Q-sepharose column (40 ml) was used and the flow
rate was increased to 4 ml/min (due to the increase in column volume) and 6 mls of
sample was injected onto the column. Six peaks were seen and are shown in Figure 4.8.
Qseph.8.Pk2 required very little NaCl to elute from the column (60mM) while
Qseph.8.Pk3 & Pk4 required 115 mM and 150 mM NaCl to elute. However,
Qseph.8.Pk5 & Pk6 required 175 mM and 450 mM NaCl to elute from the column.
Native PAGE of these two fractions revealed they contained the bands that
(Rüdiger, 1998). As some lectins require metal ions to be present for their functioning
(Sharon & Lis, 1988; Loris et. al., 1998), the buffering system was changed to m
_________________________________________________________________ 83
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
corresponded to the SO2 and SO5 proteins and no lectin activity was detected within
these fractions (refer to Table 4.1).
Figure 4.8: Large scale Q-sepharose column at pH 8.0.
The gradient used was 0-12%, 12-23%, 23-35% 35-100% 0.5 M NaCl/20 mM Tris-HCl pH 8.0
Native PAGE results showed the Qseph.8.Pk2 fraction to contain the proteins SO3 and
I. Interestingly, the Qseph.8.Pk1 fraction contained lectin activity but did
at a flow rate of 4 ml/min. Seven peaks were detected and labelled Qseph.8.Pk1-Pk7.
not show any
ands on the gel. This could be due to the levels of protein for the Qseph.8.Pk1 fraction
Lectins only constitute up
to 10% o the total pro within eeds and therefore the amount of protein that
correspo c be very lo ithin th tion a ay not be detected
on nativ s lectin isolated from e seeds of G.robusta
was dete e the total seed content. This could explain the
absence of a band on native PAGE.
The second possibility involves the overall charge of the protein. If the protein was
positively charged it would not migrate through the gel and will not be detected. It was
later found that this was not the reason for the absence of band on native PAGE. The
b
were well below the detection limits for the staining system.
f tein content s
nds to the le tin would w w e frac nd m
e PAGE. A seen in chapter 5, the th
rmined to b less than 5% of
_________________________________________________________________ 84
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
lectin was isolated (refer to Chapter 3) from the seeds of G.robusta and was identified
The GAT bioassay was used to identify the fractions containing the lectin within each
eluted fraction. Qseph.8.Pk1 & Qseph.8.Pk2 were found to agglutinate granulocytes in
assay are shown in Table 4.1. From these
preliminary results shown in Table 4.1, Qseph.8.Pk2 was used to determine the sugar
specificity of the lectin proteins. This fraction was diluted to 1:50 and tested in the
GAT and GIFT bioassays to determine the saturation point of the lectin binding to the
granulocyte receptors. This information provided the working solution for the sugar
blocking GIFT bioassay. The results of the titre are shown in Table 4.2.
obusta seeds. #
Native PAGE GAT score
as the band corresponding to SO1 on native PAGE.
the GAT bioassay and therefore contained the lectin proteins. A summary of these
results for both native PAGE and the GAT bio
Table 4.1: Native PAGE and GAT bioassay results of eluted fraction from the
large-scale purification of proteins from G.r
pH 8.0 Neat 1:2 1:4 Qseph.8.Pk1 no bands 3+ 2/3+ 2/3+ Qseph.8.Pk2 I, SO3 3+ 2/3+ 1/2+ Qseph.8.Pk3 SO1, GR1, SO4, SO5 0 0 0 Qseph.8.Pk4 SO1, GR1, SO4 0 0 0 Qseph.8.Pk5 GR1,SO4, SO5 0 0 0 Qseph.8.Pk6 GR1, SO5 0 0 0
ge of 0-4+.
#A 13% separating (with a 3% stacking gel) native gel was made and stained with Coomassie
Brilliant Blue. The GAT bioassay was graded from a ran
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Table 4.2: Saturation point on granulocytes using the Qseph.8.5.Pk2 fraction. #
Dilution GAT score CF Rel.M1:2 2/3+ .97 331:5 1+ .55 17
1:10 0 8.73 1:15 0 6.57 1:20 0 5.34 1:50 0 2.83
#The GAT was scored from e 4+ indica rong agglutina Rel. MCF value
above 4 indicated positive binding in the GIFT.
saturation for the lectin-granulocyte
inding was between 1:2 and 1:5. If the 1:15 dilution were used, it would be difficult to
disaccharides) at three different concentrations (0.1 M, 0.25 M, &
.5 M) to determine the specificity of each sample. Each sugar was incubated with the
0-4+ wher ted st tion. A
From Table 4.2, the dilution chosen for the sugar blocking GIFT bioassay for the G.
robusta lectin was 1:10 because the point of
b
distinguish the difference between a positive and negative result as this dilution gave a
Rel. MCF value of 6.57. On the other extreme, if the 1:5 dilution was used, there would
be too many binding sites to fill with sugar and effective blocking of those sites might
not occur, thus providing inaccurate results.
The sugar blocking GIFT bioassay used eight different sugars (a combination of
monosaccharides &
0
diluted lectin solution (1 part lectin & 4 parts sugar) prior to adding the mixture to the
granulocytes. The results are shown in Table 4.3.
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Table 4.3: Sugar-blocking GIFT results of Qseph.8.Pk2. †
Pk2 (1:10) Pk2 (1:10) 0.5M Sugar MCF Rel. MCF
L-fucose 0.946 6.66 Galactose 0.91 5.83 N-acetylgalactosamine 1.09 6.99 glucose 1.13 7.24 N-acetylglucosamine 1.14 7.31 lactose 0.94 6.03 maltose 0.856 5.49 mannose 0.633 4.06 No block 1.4 8.97
† All sugars are D-sugars unless otherwise indicated. The decrease in Relative MCF (and MCF)
hese results were consistent with the preliminary results obtained for the crude extract.
The method for the large-scale purification of G.robusta proteins was further modified
by increasing the pH from 8.0 – 8.5 (to increase the binding of the proteins to the
column). Figure 4.9 shows the elution pattern for the proteins when the pH is changed
(9 peaks were detected and labelled Qseph.8.5.Pk1 – Qseph.8.5.Pk9).
was detected in the mannose sample (shown in bold).
Mannose was found to block the sugar-binding site of the proteins in Qseph.8.Pk2.
T
A large number of lectins are specific for mannose/glucose, as the only difference
between the two sugars is the orientation of the OH group around C2 in the ring. The
lectin in G.robusta was found to be specific for mannose only, suggesting the
orientation of the OH group at the C2 of the sugar is important in the binding of the
sugar to the lectin.
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Figure 4.9: Large scale Q-sepharose column at pH 8.5.
The pH of the buffers were increased to 8.5 and the column was run with a flow rate of 4
ml/min using the gradient 0-12%, 12-23%, 23-35%, 35-100% 0.5 M NaCl/ 20 mM Tris-HCl pH
8.5. Nine peaks were detected and labelled Qseph.8.5.Pk1-Qseph.8.5.Pk9.
Native PAGE and GAT bioassay were used to identify and characterise the proteins
eluted using this method at pH 8.5. The samples were diluted to 1:8 for the GAT
bioassay and to 1:4 in the GIFT. Fractions Qseph.8.5.Pk1 through to Pk6 returned
positive results to the GAT and GIFT bioassay for the presence of lectins. Table 4.4
summarises these results from native PAGE and the GAT and GIFT bioassays for the
fractions eluted from the column.
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Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Table 4.4: Summary of native PAGE and bioassay results of the proteins eluted #
Native PAGE GAT GIFT
from the large-scale Q-sepharose column at pH 8.5. 13% separating gel Score Rel. MCF Rel. MCF 3% stacking gel 1:2 1:4 1:2 1:4 Crude extract All bands 3+/4+ 3+/4+ 23.61 23.16 Qseph.8.5.Pk1 Blank 2+/3+ 2+ 34.18 21.71 Qseph.8.5.Pk2 Blank 3+ 3+ 64.56 46.20 Qseph.8.5.Pk3 Blank 2+ 1+/2+ 16.08 8.92 Qseph.8.5.Pk4 I, SO1 3+ 2+/3+ 52.97 -- Qseph.8.5.Pk5 SO1,SO3, SO5 2+ 1+/2+ 42.66 24.49 Qseph.8.5.Pk6 SO1,SO4 1+ 0 27.47 21.39 Qseph.8.5.Pk7 SO1,GR1,SO4,SO5 0 0 9.87 5.51 Qseph.8.5.Pk8 GR1,SO4, SO5 0 0 14.11 9.68 Qseph.8.5.Pk9 SO4 1+/2+ 0 21.08 13.48 #Bands were stained with Coomassie brilliant blue on native PAGE. The GAT bioassay was
scored from 0-4+. A positive result is a score of 2+ or better. A Rel. MCF value greater than 4,
dicates some interaction or binding of the protein to the surface structures of the granulocyte.
H 8 (Qseph.8.Pk4). A
umber of runs were completed using the large-scale method at pH 8.5 to collect
enough material for further purification.
in
Agglutination was detected in the fractions, Qseph.8.5.Pk1-Qseph.8.5.Pk6, and all of
the eluted peaks were found to bind to the surface of the granulocytes as seen with the
GIFT results. Qseph.8.5.Pk4 contained the bands, I & SO1, which were the same as
those seen in Qseph.8.Pk2. From these results, it was concluded that the fractions
containing the lectin were Qseph.8.Pk2 and Qseph.8.5.Pk4. No agglutination was
detected in Qseph.8.5.Pk7 however, this fraction did bind to the granulocytes surface
structures detected by the GIFT bioassay. Native PAGE indicated that this fraction is
made up of mainly the GR1 protein with some minor bands (SO1, SO4 and SO5) which
was similar to the fraction 4 from the large-scale method at p
n
_________________________________________________________________ 89
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
4.3 Further Purification of G.robusta proteins
Each fraction eluted from the large-scale Q sepharose method (at pH 8.5) was further
purified using a number of different chromatography techniques. The fractions that
were further investigated were Qseph.8.5.Pk4, Qseph.8.5.Pk5/6 and Qseph.8.5.Pk7
from the large-scale method at pH 8.5. All of these fractions were positive in the GIFT
and GAT bioassays except for Qseph.8.5.Pk7, which did not agglutinate cells in the
GAT bioassay. Fraction Qseph.8.5.Pk4 and the fraction containing the
seph.8.5.Pk5/6) were the first two groups to be studied.
raphy supports and
lectrophoretic techniques. It is available in two forms, crystalline and spherical. There
are two different types of spherical CHT-type I and type II. CHT-type I has a high
finity for acidic proteins but provides better
(Q
Hydroxyapatite chromatography was used in the attempt to separate the proteins in
Qseph.8.5.Pk4. Hydroxyapatite is a form of calcium phosphate (Ca5(PO4)3OH)2) which
has been used in the separation of a number of proteins, enzymes, nucleic acids and
macromolecules (Bio-Rad Laboratories, 2001). Hydroxyapatite allows the separation of
molecules that are usually homogenous to other chromatog
e
protein binding capacity and has a high affinity for acidic proteins while the CHT-type
II resin has a lower binding capacity and af
resolution of proteins that eluted at lower NaP concentrations.
_________________________________________________________________ 90
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
4.3.1 Further purification of the GR1.Qseph and SO4/5.Qseph proteins.
A CHT II column was used to separate the SO1/2 and SO4/5 proteins. The column was
equilibrated in 10mM sodium phosphate buffer (NaP) pH 6.9 and 0.6ml of SO1/2 &
SO4/5 mixture was injected onto the column. A gradient of 0-100% 400 mM NaP pH
6.9 was used to elute bound proteins at a flow rate of 0.8 ml/min. Three peaks were
detected using UV (at a wavelength of 280 nm) and native PAGE was used to identify
these eluted peaks (Figure 4.10).
Native PAGE revealed CHTII.Pk1 to contain the SO4/5 proteins while CHTII.Pk2 and
CHTII.Pk3 both contained the SO1/2 proteins. Therefore, the SO4/5 proteins could be
separated from the SO1/2 proteins using the CHT II column. Due to the low protein
concentration, these samples were not tested for biological activity. Other workers took
on the project to isolate the lectin proteins from Qseph.8.5.Pk4 and Qseph.8.5.Pk5/6
however; their work did not result in the purification of the lectin.
_________________________________________________________________ 91
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Figure 4.10: Hydroxyapatite chromatography of the proteins SO1/2 and SO4/5
derived from the Q sepharose method.
continuous gradient was used (0-100% 400 mM Na Phosphate buffer pH 6.9) at a flow rate of
0.8 ml/min. Three peaks were detected and labelled (CHTII.Pk1-Pk3).
as further
urified using reverse phase high-pressure liquid chromatography (RP-HPLC) which
A
4.3.2 Further purification of the GR1.HPLC protein.
The final fraction, Qseph.8.5.Pk7, which contained the proteins GR1/4, w
p
separates proteins on the basis of their hydrophobic nature. The resin is made up of
silica that can contain a number of bonded phases (C-4, C-8, & C-18) that vary
depending on the hydrophobicity of the sample (C-18 is used for hydrophilic samples).
The sample is injected onto the column in water and a gradient applied using a
hydrophobic solvent such as acetonitrile to elute the proteins from the column.
_________________________________________________________________ 92
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
A Rainin C-18 analytical column was used to separate the GR1/4 proteins. The column
was equilibrated in 0.1% TFA/ Milli-Q water and acetonitrile/0.1% TFA was the eluting
olvent. A continuous gradient (0-100% acetonitrile) was used initially to determine
where the proteins would elute and this resulted in two peaks eluting at approximately
Using this information, the method was fine-tuned to establish the best conditions for
eins. A small gradient of 25-32% acetonitrile/ 0.1% TFA
ver a period of 10 minutes was found to provide effective separation of these proteins
s
25% acetonitrile/0.1% TFA (results not shown).
the separation of the two prot
o
as seen in Figure 4.11. Two peaks were seen and native PAGE showed HPLC.Pk2 to
correspond to the GR1 protein (results not shown).
Figure 4.11: RP-HPLC of GR1/4 proteins using the gradient 25-32%
The column was equilibrated at 25% acetonitrile/TFA and the gradient was increased over a
period of 10 minutes t
acetonitrile/0.1% TFA.
o 32%. Two main peaks were detected using UV at a wavelength of
and labelled Pk1 and Pk2. 280nm
_________________________________________________________________ 93
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
The method was altered to determine whether the proteins could be eluted using an
isocratic flow rate. This was important as there was a large volume of protein sample to
proce protein could be purified in a shorter tim An isoc ic flow rate
at 29% a / 0.1% FA w ed to arate fract seen he gradient
ethod (Pk1 & Pk2). The results of this method are shown in Figure 4.12.
in corresponding to the GR1 protein. This
action was concentrated using an ultra filtration stirred cell (membrane cut off of 3000
Da). The GR1 protein isolated using this method is referred to as the GR1.HPLC
protein.
ss, and the more e. rat
cetonitrile T as us sep the ions in t
m
Native PAGE was used to analyse all of the peaks that eluted from the column and the
two main peaks were labelled HPLC.Pk1 & HPLC.Pk2. The fraction labelled
HPLC.Pk2 contained a number of peaks which were the result of the protein interacting
with the resin as it was eluting under the isocratic flow rate. Native PAGE revealed the
fraction HPLC.Pk2 to contain the prote
fr
Figure 4.12: RP-HPLC using an isocratic flow rate at 29% acetonitrile/TFA 0.1%.
Two peaks were detected and labelled HPLC.I.Pk1 & HPLC.I.Pk2.
_________________________________________________________________ 94
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
4.3.3 Bioassays
Bioassays were completed on the purified GR1.HPLC protein. In both the GAT and
GIFT bioassays, no agglutination or binding was seen for the purified GR1.HPLC
protein. However, the starting material prior to RP-HPLC (Qseph.8.5.Pk7) indicated
some binding to granulocytes in the GIFT bioassay and no agglutination in the GAT
bioassay. It was proposed that the solvents used in the isolation of the GR1.HPLC
protein altered the binding site or structure of the protein, which inhibits their ability to
n experiment was set up to determine whether this was the case. The GAT bioassay
Table 4.5: GAT bioassay and the biological effects of the solvents on the crude
extract. #
Neat 1:2 1:4 1:10 1:50 1:100
bind to their specific sugar.
A
was used to determine the effects of the addition of solvents (the same solvents used for
the isolation of GR1.HPLC protein) on the crude extract. The crude extract was used
for this bioassay as it provided a strong positive result to agglutination and any decrease
in agglutination could be easily identified. The solvents (acetonitrile/0.1% TFA and
0.1% TFA/water) were diluted to 1:100 with water and added to the crude extract (a
control was also used (diluted to 1:5) to ensure the assay was working). The results are
shown in Table 4.5.
Crude extract (1:5) 4+ -- -- -- -- -- Acetonitrile/TFA 0 0 0 1+ 3+ 3+/4+
TFA/water 0 0/1+ 3+ 3+ 3+ 3+ #The bioassay is scored from 0-4+ where 4+ represents strong agglutination.
_________________________________________________________________ 95
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
No agglutination was detected for the extract in acetonitrile/TFA and TFA/water, until
the dilution reached 1:10 for acetonitrile/TFA and 1:2 for TFA/water (as seen in Table
3.5). For agglutin % or less of tr e/TFA 0% or less for
TFA/water would be required. fo runn he 4.Q m e on a
colu r the current conditio (29% ceto /TF 71% FA/w r) would
result in decreased biological activity.
.3.4 Purification of the GR1.HighQ protein
Another method was required that maintained the biological activity of the
purification process. A different anion exchange resin
ng this method will be referred to as the
ation to occur, 10 acetoni il or 5
There re, ing t GR1/ seph ixtur
mn unde ns a nitrile A, T ate
4
GR1/4.Qseph proteins during the
was used to further separate the GR1/4.Qseph proteins. A High Q column (10ml;
strong anion resin from Bio-Rad Laboratories) was made and equilibrated in 20 mM
Tris pH 8.5. Proteins were separated using a gradient of 0-30% 0.5 M NaCl (in 20 mM
Tris pH 8.5) over 10mls. Five peaks were seen and are shown in Figure 3.13. Native
PAGE was used to identify the eluted proteins. HighQ.Pk4 eluted at 110 mM NaCl and
contained the GR1 band while HighQ.Pk5 contained the SO4 band and eluted with 175
mM NaCl. The GR1 protein isolated usi
GR1.HighQ protein.
_________________________________________________________________ 96
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Figure 4.13: The separation of GR1/4.Qseph proteins using a High Q column.
The proteins were eluted from the column using a gradient of 0-30% 0.5 M NaCl in 20 mM Tris
pH 8.5 over 20 minutes. The peaks are labelled HighQ.Pk1- Pk5.
GAT and GIFT bioassays were completed on the eluted fractions from the High Q
column (HighQ.Pk1-Pk5). As determined earlier, the GR1/4.Qseph mixture provided a
negative result in the GAT bioassay. Eluted fractions were tested undiluted while the
starting material and the crude extracts were diluted to 1:10. The bioassay results are
shown in Table 4.6. Agglutination was not seen in any of the eluted fractions in the
GAT bioassay. This was to be expected, as the starting material did not agglutinate
cells. The GIFT bioassay did reveal some binding in HighQ.Pk4 (GR1.HighQ).
_________________________________________________________________ 97
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
Table 4.6: The GAT and GIFT bioassay results of the eluted fractions using High-
in. GIFT
Q res #
GAT Score Rel.MCF
Sample Neat 1:2 1:5 1:10 Neat 1:2 1:5 1:10
Crude extract 3+ 3+ 2+/3+ 2+ 31.17 26.02 20.78 17.57 Qseph.8.5.Pk7 0 0 0 0 10.39 9.09 5.01 2.63 HighQ.Pk1 0 -- -- -- 10.00 -- -- -- HighQ.Pk2 0 -- -- -- 4.49 -- -- -- HighQ.Pk3 0 -- -- -- 7.96 -- -- -- HighQ.Pk4 0 -- -- -- 6.29 -- -- -- HighQ.Pk5 0 -- -- -- 2.59 -- -- --
# + +
value indicates the level of binding of the lectin to the surface of the neutrophil.
4.4 N-terminal Sequencing
more conclusive N-terminal sequencing
results. All of the bands seen on native PAGE were sent for sequencing; however, some
of the samples did not return a sequence as their N-terminal regions were blocked with
sugars or there was very little sample present. The proteins that did return sequences
are summarised in Figure 4.14.
SO1.High Q
The GAT is scored between 0-4 where 4 represents strong agglutination. The Rel.MCF
Preliminary N-terminal sequencing results using the crude extract provided a number of
inconclusive results. The separation of proteins during the initial ion exchange
chromatography step (Q-sepharose) provided
G G D E R E F ? E D D V V T T S/ I/ P P R -- -- -- -- --
R R
GR1 "Crude" G G E E A D W (C) E D D V V T T S (C) S I P P -- -- -- --
GR1.Qseph G G E E A D (C) (C) E E D V V Q C T I V -- -- -- -- -- -- --
GR1.High Q G G E -- A D F (C) E D D V V T -- R I I/ P P ? K R ? T
P
SO4.Qseph S I P P E A D R E T D S V V V V K -- -- -- -- -- -- -- --
Figure 4.14: N-terminal sequences of proteins seen on native PAGE.
between N-terminal sequences.
Overlapping residues are shown in red.
The yellow highlighted residues indicate similar residues
_________________________________________________________________ 98
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
The samples used for the sequencing of the proteins were taken at a number of different
stages of purification. The similarities in residues are highlighted in yellow. The
SO1.HighQ sequence was obtained when the SO1 protein was purified using High-Q
resin. The GR1 “Crude” sequence is the GR1 band taken from the crude extract. The
GR1.Qseph and SO4.Qseph are the GR1 and SO4 bands seen in the fraction
Qseph.8.5.Pk7. The GR1.HighQ is the purified GR1 protein after running it down the
High-Q column. N-terminal sequencing results of the SO4 protein revealed a region
that overlapped with the sequencing results from GR1 protein (shown in red). It was
roposed that the SO4 protein was a cleaved product of GR1 protein. p
_________________________________________________________________ 99
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
4.5 Determination of the full amino acid sequence and sequence alignment
studies of the GR1.HPLC protein.
or a
d o
used to produce degenerate primers providing the full amino acid e u f the GR1
and SO4 proteins. The results and methods used to determ e th u mino a
ence are t o e
ote t
ore
h r
compared, it was found that there were differences as shown in Figure 4.15. It was
concluded from these results that the GR1 protein has under e s m o of post-
lational m e
blue.
S -- - -- -- -- -- E D
R1cDNA C V C T R S I P P R C R C T D S S V C T K C V C
R F D A F C P -- -- -- -- -- --
A fellow w ker used cDNA techniques to determine the full mino acid sequence of
the GR1 an SO4 pr teins (Clague et. al., 1999). The N-terminal sequencing data was
s q ence o
in e f ll a cid
sequ ou lined in Appendix A. Fr m these sequencing r sults, it was found that
the SO4 pr in was 100% homologous (by sequence alignmen ) with the GR1 protein
and theref it was concluded that the SO4 protein was a degraded product of the GR1
protein. W en the full amino acid sequence and the N-te minal sequences were
gon o e f rm
trans odification within the se d.
GR1cDNA M A V A K V A L M I T L M V L L F V A T L P A PGR1 N-term -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
GR1cDNAGR1 N-term
T --
A --
T -
S --
N P --
F --
G P F --
R --
P S --
GG G
G EE
EE
AA D
D WW
CC
E D
GGR1 N-term D V V T T S I P P R D R E T D S V V V V K -- -- --
GR1cDNA Y L T V P A A M R P Y C E S M A SGR1 N-term -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
Figure 4.15: Comparison between the full amino acid sequence and the N-terminal
sequencing results for the GR1 protein. The N-terminal sequencing results obtained for the GR1.HPLC protein is shown in red while the
sequencing results obtained for the SO4 protein are shown in
GR1cDNA I G S L Q S Y NGR1 N-term -- -- -- -- -- -- -- --
_________________________________________________________________ 100
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
The sequencing alignment program called BLAST (Basic local alignment search tool)
hibitors from the Bowman-Birk inhibitor family. The
equences were aligned and shown in Figure 4.16.
as a
protein when comp re the her members of the Bowman-Birk f m y ithin the
in
position and the cysteine residues located in front of the reactive site of the
o d position. I w s th re ore
ought that these residues play an important role in stabilising the structure of the
was used to determine whether there were any similarities between the amino acid
sequence of the GR1 protein and other known proteins (Altschul et. al., 1990). BLAST
(from NCBI) searches the Brookhaven Protein Database and Swissprot databases and
provides information on the level of homology of the unknown protein with a number of
known proteins. The results of this search found the GR1 protein to be similar to a
number of serine protease in
s
It w found th t the cysteine residues were highly conserved throughout the GR1
a d to ot a il . W
tryps -binding region, all but one of the cysteine residues were located in the same
chym trypsin binding region were foun to be in the same t a e f
th
proteins by forming disulfide bridges.
_________________________________________________________________ 101
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
1 10 20 30
GR1 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- G G E E A D W C E D C V C
1BBI -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D D E S S K P C C D Q C A C 1PI2 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D E Y S K P C C D L C M C IBB3_DOLAX -- -- -- -- -- -- -- -- D H H H S T D E P S E S S K P C C D E C A C IBB4_DOLAX -- -- -- -- -- -- -- -- -- H E H S S D E S S E S S K P C C D L C T C IBB_PHAAU -- -- -- -- -- -- -- -- -- -- -- -- S H D E P S E S S E P C C D S C D C IBB2_PHAAN -- -- -- -- -- -- S V H H Q D S S D E P S E S S H P C C D L C L C IBB3_SOYBN M C I L S F L K S D Q S S S Y D D D E Y S K P C C D L C M C IBB2_SOYBN -- -- -- M E L N L F K S D H S S S D D E S S D P C C D L C M C
’ P1GR1 T R
31 P1 P1 40 50 P1 60’
S I P P R C R C T D S -- -- -- -- -- -- S V C T K C V C Y L T
1BBI T K S N P P Q C R C S D M R L N S C H S A C K S C I C A L S PI-II T R S M P P Q C S C E D R I -- N S C H S D C K S C M C T R S IBB3_DOLAX T K S I P P Q C R C T D V R L N S C H S A C S S C V C T F S IBB4_DOLAX T K S I P P Q C G C N D M R L N S C H S A C K S C I C A L S IBB_PHAAU T K S I P P E C H C A N I R L N S C H S A C K S C I C T R S IBB2_PHAAN T K S I P P Q C Q C A D I R L D S C H S A C K S C M C T R S IBB3_SOYBN T R S M P P Q C S C E D I R L N S C H S D C K S C M C T R S IBB2_SOYBN T A S M P P Q C H C A D I R L N S C H S A C D R C A C T R S
61 70 80 90
GR1 V P A A M R P Y C E S M A S R F D A F C P I G S L Q S Y N --
1BBI Y P A Q C F C V D I T D -- -- -- -- -- -- F C Y E P C K P S E D D PI-II Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB3_DOLAX I P A Q C V C V D M K D -- -- -- -- -- -- F C Y A P C K S S H D D IBB4_DOLAX E P A Q C F C V D T T D -- -- -- -- -- -- F C Y K S C H N N A E K IBB_PHAAU M P G K C R C L D T D D -- -- -- -- -- -- F C Y K P C E S M D K D IBB2_PHAAN M P G Q C R C L D T H D -- -- -- -- -- -- F C H K P C K S R D K D IBB3_SOYBN Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB2_SOYBN M P G Q C R C L D T T D -- -- -- -- -- -- F C Y K P C K S S D E D
Figure 4.16: Sequence alignment of the GR1 protein with a number of protease
inhibitors from the Bowman-Birk protease inhibitor family.
Cysteine residues are boxed and the reactive site is shown by the P1-P1’ scissle bond (in blue).
The trypsin-binding site is highlighted in yellow and the chymotrypsin-binding site is shown in
red. 1, GR1 (Grevillea robusta inhibitor). 2, 1BBI (Bowman-Birk Inhibitor, Werner &
Wemmer, 1992). 3, PI-II Glycine max, Chen et. al., 1992). 4, 1BB3_DOLAX & 5,
1BB4_DOLAX (Dolichos axillaris inhibitor, Jourbert et. al., 1979). 6, 1BB_PHAAU
(Phaseolus aureus (mung bean) inhibitor, Zhang et. al., 1982). 7, 1BB2_PHAAN (Phaseolus
angularis (adzuki bean) inhibitor, Kiyohara et. al., 1981). 8, 1BB3_SOYBN & 9,
1BB2_SOYBN (Glycine max, Joudrier et. al., 1987).
_________________________________________________________________ 102
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
The Bowman Birk inhibitor contains 14 cysteine residues that form 7 disulfide bridges
while the GR1 protein has only 10 cysteine residues that could form 5 disulfide bridges.
The disulfide-bonding pattern for the Bowman-Birk inhibitor is shown in Figure 4.17.
However on closer inspection of the sequence alignment of the two proteins, it was
thought that the GR1 protein might form 3-5 disulfide bonds due to the positioning of
these residues (Figure 4.17).
1 10 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 30GR1 -- G G E
1BBI D D E S
E A D W C E D C V C T R S I P P R C R C T D -- -- -- --
S K P C C D Q C A C T K S N P P Q C R C S D M R L N
40 31 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 50 60
1BBI S C H S A C K S C I C A L S Y P A Q C F C V D I T D -- -- -- --
1BBI -- -- F C Y E P C K P S E D D K E N
The scissle bond for both trypsin and chymotrypsin (P1 & P1') are highlighted in yellow. The disulfide
ridges found in the Bowman-Birk inhibitor are shown in different colours. The conserved cysteine
within this protein.
3' and P4' of the trypsin binding region were
und to be conserved within the Bowman-Birk family and the GR1 protein. A
threonine at the P2 site is highly conserved throughout all Bowman-Birk inhibitors and
onserved throughout
the Bowman-Birk inhibitor family and has been shown to be important in the
GR1 S -- -- S V C T K C V C Y L T V P A A M R P Y C E S M A S R F
61 70
GR1 D A F C P I G S L Q S Y N -- -- -- --
Figure 4.17: Position of the disulfide bridges of the Bowman-Birk inhibitor and
sequence alignment with the GR1 protein.
b
residues in the GR1 protein are also coloured providing an insight into the possible disulfide bond pattern
Within the trypsin binding region, the P1' residue (highlighted in yellow in Figure 4.18)
is conserved throughout the Bowman-Birk family and this was also found to be true for
the GR1 protein. Residues at position P
fo
this was also true for the GR1 protein. The threonine residue is c
_________________________________________________________________ 103
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
functioning of the inhibitor. Therefore, the presence of this residue in the same position
ight be important for the function of this protein within the GR1 protein suggests it m
(McBride et. al, 1998).
The reactive site for chymotrypsin contains a serine in the P1' position and a conserved
serine. It is possible that the P1-P1' region of the GR1 protein functions in a different
conserved throughout the family and this was also seen in the GR1 protein. Like the
leucine residue at P1. The GR1 protein does not have the conserved serine residue in
the P1’ position, instead, it contains a threonine residue, which could substitute for the
manner to other Bowman-Birk inhibitors. At position P3', the residue proline is
trypsin-binding region, this position may be important in forming the turn between two
β-sheet structures within the GR1 protein.
_________________________________________________________________ 104
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
4.6 Mass Spectroscopy (MS) of the GR1.HPLC protein
n was injected (Figure 3.18). Utilising the
ass spectrometry software, these 4 ion peaks were related to each other (charged
pecies are shown in red in the Figure 4.18) and the molecular weight of the GR1.HPLC
protein was calculated to be 6669 Da.
Mass spectrometry was used to determine the molecular weight of the GR1.HPLC
protein. Mass spectrometry is a useful tool in providing structural information about the
molecule. The principle behind this form of spectroscopic technique is that a small
amount of sample is bombarded with a high-energy electron beam resulting in a
molecular ion (M+) being produced. The instrument is set up to detect the mass to
charge ratio of the molecular ions, which therefore determines the molecular weight of
the molecule.
A single quadruple electrospray mass spectrometer (Fisons instruments) was used to
determine the molecular weight of the GR1 protein has a detection limit of 1 charge per
2000 Da. SDS PAGE and gel filtration chromatography suggested the GR1 protein to
have an approximate molecular weight of 7000 Da. This spectrometer could still be
used to predict the molecular weight of the protein by detecting the multiply charged
species of the protein.
Positive electrospray was used and a number of ion peaks were detected on the
spectrometer after the GR1.HPLC protei
m
s
_________________________________________________________________ 105
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________ Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
___________________________
Figure 4.18: Positive electrospra
There are 4 ion peaks that correspo
calculated to be 6669Da using the M
The program Peptide Mass in ExP
of the cDNA encoded GR1 prot
values obtained were in the redu
7285.31 Da. This value is very
GR1.HPLC protein (6669 Da).
some proteolysis. A number of
entered into the Peptide Mass p
actual value for the GR1.HPLC
(LQSYN) at the C-terminus was
mass of 6679.67 Da which w
GR1.HPLC protein. It should b
GR1.HPLC protein (without the
GR1.HPLC protein contains 10 cy
___________________________
[M]/6+
___________
the GR1
y of the GR1
nd to
assLynx softw
ASy was use
ein (Wilkins
ced form and
different from
It was therefo
residues were
rogram until
protein. It w
removed fro
as 10 Da of
e noted that
5 residues) w
steine residu
___________
[M]/5+
________________
.HP
.HPLC protein.
LC protein. The
are provided with the
d to calculate the th
et. al, 1997; Wilkin
the average mass
the experimental v
re concluded that t
subtracted from th
a value was obtain
as found that whe
m the sequence it
f the actual mass
the theoretical ma
as determined in a
es and therefore has
________________
[M]/4+
[M]/7+
___________ 106
molecular weight was
spectrometer.
eoretical mass and pI
s et. al, 1998). The
was calculated to be
alue obtained for the
he protein undergoes
e C-terminus and re-
ed that matched the
n the last 5 residues
provided an average
determined for the
ss calculated for the
reduced state. The
the potential to form
___________ 106
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
5 disulfide bonds. If all of the cysteine residues were involved in forming disulfide
the
ctual mass of the protein and thus giving a mass of 6679 Da. Therefore, all cysteine
nvolved in forming disulfide bridges and the
protein undergoes post-translational modifications at the C-terminus within the seed.
bonds and the protein was fully reduced, an additional 10 Da would be added to
a
residues within the GR1.HPLC protein are i
_________________________________________________________________ 107
Chapter 4 Purification of the GR1.HPLC protein ____________________________________________________________________________________
4.7 Conclusion
A number of different proteins were identified in the crude extract from G.robusta. A
large-scale chromatography method was established which partially separated these
proteins into three different groups. Two of these groups of proteins were reactive in
both the GIFT and GAT bioassays while the other group was only reactive in the GIFT
bioassay. Initial work on the isolation of the lectin protein was started and was taken
over by co-workers. The group of proteins that was reactive only in the GIFT bioassay
contained the GR1/4.Qseph proteins. These proteins were successfully separated using
two different methods, RP-HPLC and ion exchange chromatography. The GR1.HPLC
d had the 5 C-terminal residues removed by post-
anslational modification. The full amino acid sequence was determined and sequence
protein was N-terminally sequenced and this information was used to generate
degenerative primers to determine the full amino acid sequence of the protein. Mass
spectrometry determined the molecular weight of the GR1.HPLC protein to be 6669 Da.
The cDNA encoded GR1 protein was found to contain 5 disulfide bridges (identified by
comparing the molecular weights of the theoretical and experimental masses using the
Peptide Mass program in ExPASy) an
tr
alignment studies have shown the GR1 protein to belong to the Bowman-Birk
subfamily of serine protease inhibitors.
_________________________________________________________________ 108
Chapter 5 Purification and Characterisation of the GR1.GF protein ______________________________________________________________________
Chapter 5 Purification and characterisation of the GR1.GF
protein.
_________________________________________________________________ 109
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
Chapter 5 Purification & characterisation of the GR1.GF protein
he full amino acid sequence of the GR1.HPLC protein suggested the protein to be
5.1 Introduction
A serine protease inhibitor was isolated from G.robusta using ion exchange
chromatography and RP-HPLC. Serine protease inhibitors are one of four different
protease inhibitors identified in nature. The characterisation of these inhibitors is based
on the amino acids involved within the reactive site (refer to section 1.9 for more
information).
T
cleaved during the extraction process (leaving a minor contaminant seen on native
PAGE) (section 4.5). This chapter will outline the extraction of the serine protease
inhibitor from the seeds of G.robusta without proteolysis, the isolation and
characterisation of the GR1 protein.
_________________________________________________________________ 110
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
5.2 Extraction of the proteins from the seeds of G.robusta.
s of proteolysis during the extraction stage, protease inhibitors
ferent protease inhibitors to ensure all proteases found naturally in seeds
would be inactive. A commercially available mixture of protease inhibitors was used
which contained the following protease inhibitors: 4-(2-aminoethyl)benzenesulfonyl
fluoride (AEBSF), trans-epoxysuccinyl- L-leucylamido(4-guanidino)butane (E-64),
bestatin, leupeptin, aprotinin and sodium EDTA. PMSF was also added to the buffer.
Ground seeds were soaked in the extraction buffer containing protease inhibitors (TBS
+ PI’s) overnight at 4ºC. Ammonium sulfate was used as before (refer to section 3.2) in
two stages to precipitate the proteins from solution. Once precipitated, the proteins
s
the
and corresponding to SO4 was the result of protein degradation.
To minimise the chance
(PI’s) were added to the TBS buffer. The extraction buffer contained TBS and a wide
range of dif
were resuspended in milliQ water and initially dialysed in milliQ water (2 change
every 30 mins) and then against TBS + PI buffer pH 7.8 (2 changes every 30 mins) to
remove excess ammonium sulfate.
Native PAGE was used to visualise the extracted proteins. The results are shown in
Figure 5.1. Native PAGE revealed an absence of the band in the crude extract prepared
with protease inhibitors, which corresponded, to SO4. These findings supported the
initial findings from the N-terminal sequencing and cDNA sequencing data that
b
_________________________________________________________________ 111
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
_________________________________________________________________ 112
Figure 5.1: Native PAGE of the crude extracts processed without and with
protease inhibitors.
The gel was stained with Coomassie Brilliant Blue. Lane 1: crude extract processed with PBS
pH 7.3; Lane 2: crude extract processed with TBS + PI’s pH 7.8. The arrow shows the absence
of the SO4 band in Lane 2.
5.3 Purification of the GR1.GF protein from the crude extract
The crude extract (containing protease inhibitors) was further purified using gel
filtration chromatography. A superdex 75 prep grade gel matrix was used to separate
the proteins from the crude extract. Superdex 75 is made up of highly cross-linked
porous agarose beads and covalently bound dextrin (Pharmacia LKB Biotechnology,
1991). The combination of dextran and cross-linked agarose stabilises the matrix,
physically and chemically. The pore size for the superdex 75 resin is in the range of
3000 – 70 000Da, which will provide adequate separation of the proteins found within
G.robusta.
A 20cm superdex 75 column (ID of 2.5cm) was made and equilibrated with TBS + PI’s
pH 7.8. Calibration markers were used to determine whether the column was packed
evenly and provided information on the approximate molecular weight of the eluted
proteins. The proteins used to calibrate the column were Blue dextran (Mr = 2000000
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
Da), bovine serum albumin (BSA) (66000 Da), carbonic anhydrase (29000 Da),
cytochrome (12400 Da) and aprotini Da).
The approximate molecular weights of eluted pro s were de paring
the ratio of /Vo of the unknown proteins against the Ve/Vo of the standard proteins.
The void volume of the column (Vo) was identified by blue dextrin (Mr of 2000000 Da)
while the r inder of the standard teins, eluted at specific volumes (Ve). A
eights of the unknown proteins were determined by plotting their Ve/Vo values onto
c n (6500
tein termined by com
Ve
ema pro
calibration curve was created where the logarithms of the molecular weight of the
standard proteins were plotted against their respective Ve/Vo values. The molecular
w
this graph.
One ml of crude extract (prepared with protease inhibitors) was injected onto the
column at a flow rate of 1.5ml/min and the column was run in 100% extraction buffer
(+ protease inhibitors) pH 7.8. Five peaks were detected (Figure 5.2) and native PAGE
(Figure 5.3) identified the proteins within each eluted fraction.
_________________________________________________________________ 113
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
Figure 5.2: Gel filtration chromatography of the crude extract (containing
rotease inhibitors).
Five peaks were detected and labelled F1-F5. The flo ate wa . m and one ml of
crude extract was injected t h .
p
w r s 1 5 l/min
on o t e column
Figure 5.3: Native PAGE of eluted fractions from gel filtration chromatography.
A 13% separating gel (with a 3% stacking gel) was made and stained with Coomassie Blue.
pH 7.8. Lanes 3-7: eluted fractions from gel filtration chromatography labelled F1-F5.
Lane 1: crude extract prepared with PBS pH 7.3. Lane 2: crude extract prepared with TBS + PI
_________________________________________________________________ 114
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
Native PAGE showed the F2 fraction to contain all of the corresponding bands (SO1-
SO5). PAGE also revealed Fraction (F3) to contain a single band and this band was
thought to correspond to the GR1 protein. Due to a single band being present on the
Native PAGE, no additional purification experiments were applied to this fraction.
5.4 Bioassays
he crude extract was prepared with and without protease inhibitors and the fractions
ry
little binding to granulocytes and thus a negative result.
T
eluted from the gel filtration column were tested for lectin activity. The results from the
GAT and GIFT bioassays are shown in Table 5.1. The GAT bioassay of the crude
extract containing protease inhibitors indicated the activity of the lectin was maintained
when the proteins were extracted using protease inhibitors in the buffer. The GIFT
bioassay was not as conclusive as the GAT bioassay as the relative MCF value for the
crude sample prepared with protease inhibitors was below 4, suggesting none or ve
_________________________________________________________________ 115
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
Table 5.1: GAT and GIFT bioassay results.
GAT GIFT Dilution Score Rel. MCF Crude extract in Neat 2+ 16.60 PBS 1:2 1+ 14.47 Crude extract in TBS + PI's Neat 4+ 4.68 1:2 3+ 3.23 F1 N 3+ 17.48 eat 1:2 2+ 15.53 F2 Neat 0 6.64 1:2 0 6.67 F3 Neat 0 3.67 1:2 0 2.71 F4 Neat 0 3.26 1:2 0 3.25
The bioassay tested both crude extracts (i.e. with and without protease inhibitors) and the
fractions that eluted from the gel filtration chromatography. The GAT bioassay was scored
from 0-4+ and a Rel. MCF value above 4 indicates binding in the GIFT bioassay.
The lectin was identified in fraction 1 (F1) as seen in Table 5.1, however, no significant
not mean the lectin is
protein content within the seed and therefore, only a small amount of lectin would be
purified. The GIFT bioassay confirmed the presence of the lectin within this fraction by
porting a high relative MCF value. Agglutination was not seen in the remainder of the
bands were detected on native PAGE. The absence of bands does
not present but rather the total concentration of the lectin within this sample was too low
to be detected by commercial staining. Lectins only constitute up to 10% of the total
re
fractions and the GIFT bioassay revealed some granulocyte binding in fraction 2 (F2).
The peak containing the GR1 protein (F3) did not agglutinate the granulocytes in the
GAT nor was there any evidence of the proteins binding to granulocytes in the GIFT
assay. Therefore the GR1 protein purified using gel filtration chromatography did not
contain any lectin-like properties and will be referred to from this point on as the
GR1.GF protein.
_________________________________________________________________ 116
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
5.5 N-terminal sequencing of the GR1.GF protein
The GR1.GF protein was N-terminally sequenced to determine whether it was the same
protein as the one purified using reverse phase chromatography (section 3.4.2). The
sequencing results are shown in Figure 5.4. The N-terminal sequencing results for the
GR1.GF protein revealed similarities with the previously identified GR1.HPLC and
GR1.HighQ proteins. Differences were seen between the three N-terminal sequences.
The GR1.GF protein was found to have a cysteine residue instead of a glycine at
position 2 and an asparagine was found in the place of an aspartic acid at positions 6 &
11 (shown in red in Figure 5.4).
GR1.HPLC G G E E A D W (C) E D D V V T T S (C) I P GR1.HighQ G G E E A D F (C) E E D V V T C T I -- --GR1.GF G (C) E E A N W (C) E E N V V T T -- -- -- --GR1 (full sequence) G G E E A D W C E D C V C T R S I P P
Figure 5.4: N-terminal sequencing homology of the eluted GR1 proteins.
Amino acids are shown using the one letter code. Conserved regions are highlighted (in yellow)
and amino acid residues that differed from the previously determined N-terminals are shown in
red. The full amino acid sequence (derived from cDNA techniques) was used to compare the
N-terminal sequences.
The cysteine residue in the GR1.GF sequence is in brackets as this residue is destroyed
during the sequencing process. To obtain an N-terminal sequence, the amino acid
residues of the protein are hydrolysed and this often results in the modification of these
residues. The addition of strong regents such as hydrochloric acid has been found to
deaminate the amino acids asparagine and glutamine into their respective acids
(Davidson, 1997). Therefore, it is difficult to determine which residue is an aspartic
acid or an asparagine.
_________________________________________________________________ 117
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
The N-terminal sequences did not differ from the sequence derived from full sequence
derived from DNA techniques. The regions that are homologous with the full sequence
are highlighted in yellow in Figure 5.4. Therefore, N-terminal sequencing provided a
good and reasonably accurate prediction on the amino acid sequence for the GR1
protein.
The three-boxed peak values are charged species of the same protein. These species
exist because the mass spectrometer measures the ratio between the molecular weight or
mass and the charge of the protein. The mass spectrometer used in this experiment was
the same one used to identify the mass of the GR1 protein isolated using HPLC
(GR1.HPLC; refer to section 4.7 for more details). Key ion peaks that corresponded to
the HPLC purified GR1 protein were the same as those seen in Figure 5.5.
5.6 Mass Spectroscopy of the GR1.GF protein
The GR1.GF protein had a molecular weight of 6669 Da, which was determined using
mass spectroscopy. The GR1.HPLC protein also had a molecular weight of 6669 Da.
The mass spectrum for the GR1.GF protein is shown in Figure 5.5. Despite the
GR1.GF protein N-terminal sequence differing by two amino acids, the calculated
molecular weights for both proteins were the same. It was therefore assumed that the
two proteins isolated by different chromatography methods were the same proteins.
_________________________________________________________________ 118
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
_________________________________________________________________ 119
btained for the GR1.GF prFigure 5.5: Mass spectrum o otein.
Positive electrospray of the sample reveals multiple ion peaks that equate to the molecular
weight of the protein. The boxed peaks show the related peaks in this trace.
5.7 Serine protease inhibition assays
Protease inhibitors that belong to the Bowman-Birk inhibitor family are able to
inactivate both the trypsin and chymotrypsin proteases (independently). The trypsin-
binding site of the GR1 protein was very similar with the protease inhibitors from the
Bowman-Birk family and there were some regions of homology in the chymotrypsin-
binding region. An inhibition assay was set up by a co-worker at the ARCBS to
determine whether the GR1 protein (using the GR1.GF sample) belonged to the
Bowman-Birk family by inactivating trypsin and chymotrypsin (Clague, 2001).
[M]/5+
[M]/6+
[M]/4+
_________________________________________________________________ 119
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
It was found that the GR1.GF protein inactivated both trypsin and chymotrypsin and
was therefore c ember of the Bowman-Birk family. The KI values for
the inactivation of trypsin and chymotrypsin were 1.37 x 10-9 M and 4.8 x 10-9 M
respectively (Clague, 2001; Appendix B). On comparison with the Bowman-Birk
protease inhibitor (Werner & Wemmer, 1991), where the KI values for trypsin and
chymotrypsin were 5 x 10-9 M and 5.2 x 10-9 M respectively, the GR1.GF protein was
ion of trypsin (Figure B1-
A) does not fit the data points on the graph. This could explain why the inhibitory
haracterised as a m
found to be similar in its ability to inactivate these proteases.
A fellow co-worker, as a part of her Masters research, generated the inhibition curves
for the GR1.GF protein (seen in Figure B1 {Appendix B}) (Clague et. al., 1999, 2001).
It should be noted that the curve corresponding to the inactivat
results obtained for trypsin were lower than that seen for the Bowman-Birk inhibitor.
_________________________________________________________________ 120
Chapter 5 Purification and Characterisation of the GR1.GF protein _____________________________________________________________________________________
5.8 Conclusion
The preparation of the crude extract using protease inhibitors revealed an absence of the
protein band SO4 on native PAGE. These results supported the initial sequencing
results that the SO4 protein was a degradation product of the GR1 protein. Using gel
filtration chromatography, the GR1 protein was purified from the crude extract
containing protease inhibitors (labelled GR1.GF). Bioassays ensured the crude extract
prepared with protease inhibitors maintained lectin activity and was located in the first
fraction after gel filtration chromatography (GF.F1). The GR1.GF protein did not
agglutinate cells in the GAT bioassay nor did it bind to the granulocytes in the GIFT
bioassay. N-terminal sequencing of the GR1.GF protein showed differences in only two
amino acids when compared with the other GR1 proteins (GR1.HPLC & GR1.HighQ).
Mass spectrometry determined the molecular weight of the GR1.GF protein to be 6669
Da, which was identical to the results obtained for the GR1.HPLC and GR1.HighQ
proteins.
_________________________________________________________________ 121
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Chapter 6 NMR assignment of the GR1 protein from
Grevillea robusta
________________________________________________________________________________ 122
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Chapter 6 NMR assignment of the GR1 protein from Grevillea
robusta
6.1 Introduction
The GR1 protein was purified and characterised using HPLC and gel filtration
chromatography, mass spectroscopy, N-terminal sequencing and PAGE as described in
Chapters 3 & 4. Functional assays (Clague et. al., 1999) showed the GR1 protein could
hibit both trypsin and chymotrypsin independently and the Ki values were
in the inhibitory effects for both the GR1 protein and Bowman-Birk
hibitor could suggest a similar structure/function relationship. However, sequence
alignment showed a number of differences between the two proteins especially within
the proposed chymotrypsin-binding site. Key residues involved in forming the reactive
site towards chymotrypsin within the GR1 protein were C-X-C-X-L-T/S-X-P-A-X,
where L and T/S correspond to the P1-P1′ of the scissile bond.
in
comparable to those obtained for the Bowman-Birk inhibitor.
The Bowman-Birk inhibitor (BBI) is a member of the serine protease inhibitor family
and has been described as a double-headed protein that inhibits both trypsin and
chymotrypsin independently. It is a 71 amino acid protein that contains 14 cysteine
residues that form 7 disulfide bridges. It was first isolated from soybeans over 40 years
ago and to date, a large number of proteins isolated from plants have been characterised
as members of the Bowman-Birk serine protease superfamily (Birk, 1985).
The similarities
in
________________________________________________________________________________ 123
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Within the Bowman-Birk inhibitor, the positioning of the cysteine residues plays an
important role in stabilising the overall structure of the protein through the formation of
disulfide bridges. These bridges aid in the formation of the antiparallel β-sheet and
turn structures seen for the trypsin and chymotrypsin binding sites. In the
chymotrypsin-binding region of the GR1 protein, the cysteine residues are not
conserved relative to the Bowman-Birk inhibitor and therefore the disulfide-bonding
pattern will be different, and could result in a different structure. Due to the similar
inhibitory effect of the GR1 protein and differences within the chymotrypsin-binding
site, the structure of the GR1 protein was determined using NMR spectroscopy. This
chapter will briefly discuss the basic principles behind protein NMR spectroscopy and
outline the steps used to assign the GR1 structure.
________________________________________________________________________________ 124
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
6.2 NMR Spectroscopy
The protein samples were dissolved in 18% CD3CN /H2O pH 3.5 and the spectra was
collected using a Varian INOVA 600MHz spectrometer at both 303 and 288 K. These
conditions were chosen because the structure of the Bowman-Birk inhibitor was solved
using NMR spectroscopy under these conditions (Werner & Wemmer, 1991). One
dimensional homonuclear and 2D homonuclear and heteronuclear experiments, DQF-
COSY, TOCSY, NOESY & HSQC were acquired to provide data for the assignment of
the protein. Coupling constants could be measured using DQF-COSY spectra.
6.2.1 One dimensional NMR experiments
One-dimensional experiments were used to optimise the experimental conditions so the
experiment would yield the best dispersion of peaks in the amide region. In order to
achieve this, a number of different experimental conditions (including temperature and
pH) were trialed to provide the best peak dispersion. Figure 6.1 shows an example of
the amide region of the 1D spectrum obtained for the GR1 protein.
________________________________________________________________________________ 125
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
________________________________________________________________________________ 126
Figure 6.1: 1D spectrum of the amide region for the GR1 protein.
One-dimensional and 2D experiments (DQF-COSY and TOCSY) were acquired on
both GR1 proteins (GR1.HPLC and GR1.GF) at 303K. The amide region of the 1D
spectrum showed narrower peaks for the GR1.HPLC when compared with the GR1.GF
sample and this is shown in Figure 6.2. The broadening of the amide 1D peaks in the
GR1.GF sample was evident and could be due to protein aggregation or due to a
number of impurities within the sample. The 2D experiments confirmed the presence
of aggregation and impurities (in the form of additional peak patterns) within the
GR1.GF sample (results not shown). However, the solution structure of the GR1
protein was determined using the GR1.GF sample as this sample was biologically
active.
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Fi
(B
6.2
To
ac
4
sta
__
A
g
)
.
q
st
g
__
B
ure 6.2: The 1D NMR spectra for the amide region of the (A) GR1.HPLC and
GR1.GF proteins
2 Two dimensional NMR experiments
sequentially assign the GR1 protein, a number of 2D NMR experiments were
uired these included the DQF-COSY, TOCSY and NOESY experiments. There are
ages within a 2D experiment; the preparation, evolution, mixing and detection
es as seen in Figure 6.3 (Wüthrich, 1986). The preparation stage involves a delay
____________________________________________________________________________ 127
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
time that allows the nuclei to establish thermal equilibrium. At the end of this stage,
ne or morel rf pulses are applied to create the desired coherence.
Figure 6.3: The schematic representation of the NOESY experiment.
The P1, P2 & P3 are the pulses; t1 is the evolution time; τm is the mixing time; the FID is
ransform (FT) is applied to the time
omain data, which produces the 2D-frequency spectrum.
o
t2 t1
P1 P2
Delay
P3
τm
Preparation Evolution Mixing Detection
recorded during t2.
During the evolution stage (defined by the evolution time t1), coherence transfer is
achieved where the nuclei may adopt a different spin state and it is these differences
that allow the chemical shift values for the individual spins to be identified or labelled.
One or several rf pulses are applied in the mixing stage over a period of time denoted as
the mixing time (τm). The detection stage results in the free induction decay (FID)
being acquired and stored as time domain data. Repeating the same experiment a
number of times while increasing the evolution time after each experiment creates the
second dimension. A two-dimensional Fourier T
d
________________________________________________________________________________ 128
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
6.2.2.1 Correlated spectroscopy (COSY) and Double quantum filtered COSY (DQF-
COSY)
COSY is the simplest of the homonuclear 2D NMR experiments and is used to identify
pairs of protons involved in scalar coupling. The experiment is made up of two 90°
pulses (P1 & P2), separated by an evolution time t1 (Figure 6.4A). This experiment
was not used to assign the GR1 protein. Instead the Double Quantum Filtered COSY
(DQF-COSY) experiment was used which provided better data for the assignment of
the protein since it removes any singlets from the acquired data.
The DQF-COSY is an extension of the COSY experiment where it involves an
shor
r the (A) COSY and (B) DQ
(A) P1 & P2 are 90° pulses separated by the evolution time (t
mixing stage is shown by ∆. The FID is recorded during t2 in bo
DQF-COSY spectra are used to generally detect spin-spin
constants in individual amino acid residues in a protein, a
provide the dihedral angle restraints for structure calculati
additional 90° pulse (P3) in the mixing phase of the experiment that is separated by a
t delay (∆) from P2 (Figure 6.4B). The additional pulse converts the double
quantum coherence into single quantum coherence prior to detection.
t1
Prep Evolution Mix Detect
3
Prep E
Figure 6.4: Pulse sequence fo
_________________________________________________________
t1
F-COSY experi
1). (B) The short d
th experiments.
3JNH-Hα and 3JHα-H
nd these coupling
ons. The HN-Hα
volution Mix
_________________
t2
P1
P2t2
P1
P2 P∆
A
Bments.
elay in the
β coupling
constants
region of
Detect
______ 129
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
DQF-COSY spectra for the GR1 protein is shown in Figure 6.5. In theory, all of the
HN-Hα connectivities of the protein should be seen within this region. For GR1, not
all of the HN-Hα peaks were observed and this was due to a number of overlapping
peaks in the TOCSY/ NOESY spectra. Alternatively, peaks could be absent from the
spectra if the peaks of interest are located around the same frequency as the water that
is being suppressed.
Figure 6.5: The HN-Hα region of the DQF-COSY experiment for the GR1
.2.2.2 Total correlation spectroscopy (TOCSY)
he TOCSY experiment allows through bond connectivities between protons to be
the second pulse with a sequence called
MLEV-17 or spin lock (Bax & Davis, 1985), during the mixing stage of the experiment
protein.
6
T
determined that are not restricted to 2 or 3 bonds. The TOCSY experiment differs
from the COSY experiment by replacing
________________________________________________________________________________ 130
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
(Figure 6.6). This MLEV-17 sequence causes the magnetisation to become “spin-
locked” in one axis, which results in all spins in a coupled system to have the same
Figure 6.6: The pu
6.2.2.3 Nuclear Ove
The spectra obtained
the protein to be det
This experiment ide
less than 5 Å. The
time (τm), which all
diagram of the NO
relaxation, the magn
dependent on the l
frequency. Once the spin lock is removed and the FID is collected, the cross peaks are
detected for all spins in their respective coupled system such as the side-chain protons
in an amino acid.
The four stages are s
during t2.
n
___________________
P1
lse sequence for
rhauser Enhanc
from the homo
ermined by visu
ntifies the proto
NOESY exper
ows the pairs of
ESY experime
etisation of the
ength of the m
hown. The evolu
n
________________
M
2
t1the TOCSY exper
ement spectroscopy
nuclear NOESY ex
alising through spa
ns that are in close
iment contains thre
protons to undergo
nt is shown in Fi
protons is transferre
ixing pulse and th
tion time is represen
___________________
t
LEV-17
n
Preparatio Evolutio Mixing Detectioiment.
(NOESY)
periment allows the sequence of
ce connectivities of the protons.
proximity to each other, usually
e pulses (P1-P3) and a mixing
cross-relaxation. A schematic
gure 6.7A. During this cross
d and the rate of this transfer is
e size of the molecule being
ted by t1 and the FID is recorded
__________________________ 131
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
investigated (Roberts, 1993). Figure 6.7B is an example of the HN-HN region of the
NOESY spectrum obtained for the GR1 protein.
Figure 6.7 he (A) sc
the HN-HN region of t
(A) P1, P2 & P3 are rf pu
The HN-HN region of the
B
n
: T
_______________________
P1
hematic pulse s
he NOESY spec
lses applied. The
NOESY spectrum
n
________________
P2
eq
tra for the
mixing time
for the GR1
m
uence for
___________
P3
t1
τGR1 prot
is shown b
protein wi
the NOE
__________
t2
Preparatio Evolutio Mixing DetectionA
ein.
etween P2 & P3 by τm. (B)
th a mixing time of 200ms.
SY experiment and (B)
____________________ 132
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
6.3 The 1H NMR Assignment of the GR1 protein
6.3.1 Solvent suppression
The NMR assignment of the GR1 protein was achieved by using a combination of
DQF-COSY, TOCSY and NOESY experiments. Using the full amino acid sequence of
the protein derived from cDNA techniques and by following the approach outlined by
Roberts (1993), the spin systems of the individual amino acids within the protein were
on
pressing water. It involves a
continuous irradiation at a low power rf at the same frequency of the solvent peak
during the relaxation delay (Roberts, 1993) as shown in Figure 6.8A. In this work, the
presaturation sequence was used in the DQF-COSY experiments.
assigned. The majority of the protein structures determined by NMR spectroscopy are
dissolved in H2O or H2O/D2O mixtures that allow the researcher to study the structure
of the protein in solution. However, the proton peak for water is many times more
intense than the protein’s protons (approximately 10 000 times) and therefore this
signal needs to be suppressed. To overcome this, a number of solvent suppressi
sequences have been developed to reduce the intensity of the water protons and allow
the proteins’ protons to be visualised.
Presaturation is the easiest and effective way of sup
________________________________________________________________________________ 133P1
P2 P1 P2 A B
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Figure 6.8: Schematic representation of the water suppression sequences for (A)
(A) The long low power rf pulse applied during the relaxation delay is shown in P1. P2
corresponds to the 90° pulse applied after this delay. (B) P1 corresponds
presaturation and (B) WATERGATE.
to a non-selective 90°
ulse; P2 is the selective 180° pulse or the pulse trains targeting water. G1 & G2 correspond to
the gradient pulses that are used to suppress the solvent.
Water suppression using gradient tailored excitation or WATERGATE is the second
sequence applied to suppress the water peak in the protein NMR sample. The water is
suppressed by using two short gradient pulses of the same amplitude, separated by a
selective 180° rf pulse (Figure 6.8B). Prior to the first gradient pulse, a non-selective
90° rf (P1) is applied which results in all of the coherences being excited. These
coherences are dephased by the first gradient pulse (G1) but can be rephased by the
second gradient pulse (G2) only if they are flipped by 180° from the selective rf pulse
2) (Sklenář et. al., 1993). During this stage, application of the second gradient pulse
ing the initial WATERGATE sequence has been improved (Liu et
l; 1998). The selective 180° rf pulse has been replaced by the addition of a number of
different pulse trains, denoted W, that consist of 3, 4, or 5 pulses (Sklenář et al., 1993;
p
(P
(G2) results in the water signal being further dephased. By the time the receiver
acquires the data, there is little or no water signal observed (Sklenář et. al., 1993; Liu
et. al., 1998). The WATERGATE sequence was used to remove the water in the
TOCSY and NOESY 2D experiments in this work.
Water suppression us
a
________________________________________________________________________________ 134
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Liu, 1998). The pulse train used in this work was the 3-9-19 or W3, where 6 pulses are
applied during P2 (Figure 6.8B). The W3 is made up of the sequence 3α-τ-9α-τ-19α-τ-
19α-τ-9α-τ-3α where 26α = 180° pulse where τ was the time between the pulses, which
govern the width of the excitation region. These modifications to the original
WATERGATE sequence improved the suppression of the resulting data by narrowing
the suppressed region, ie the water, and allowing the proteins peaks to be observed (Liu
et al., 1998). There are several other methods of water suppression but since they were
not used in this work, they will not be discussed.
6.3.2 Spin system identification
R1 protein was to
efine the chemical shifts that correspond to each proton. The random coil chemical
shift values obtained by Wüthrich (1986) were used only as a guide in the assignment
f the amino acid spin systems of the GR1 protein.
+
groups are located in section A and the Hα to side-
chain connectivities are found in section C.
Spin system assignments were made using the data collected from the DQF-COSY and
TOCSY experiments. The first stage in the assignment of the G
d
o
The 2D spectrum was divided into 4 regions where each region provided information
for the assignment of the protein as seen in Figure 6.9. The HN to side-chain cross
peaks is found within section B. The aromatic protons, the NH3 of lysine and the NH
groups of arginine and amide NH
________________________________________________________________________________ 135
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
__________
Figure 6.
The four s
obtained fr
Many am
have more
experimen
spin syste
Roberts (1
Glycine w
formation
peaks eac
were dete
experimen
A
_
9:
e
o
in
t
m
9
o
h
c
t
B
_____________________________
An example of a 2D spectru
ctions are boxed in blue and lab
m the GR1 protein.
o acids have unique spin syst
complex spin systems and req
, to assign them. The assignm
s for each individual amino
93).
as identified in the Hα-HN
f an AMX spin system. The t
containing 4 antiphase comp
ted within the DQF-COSY sp
s such as the TOCSY and NO
C
B
________________________________________ 136
m.
elled A-C in red. This is the TOCSY spectrum
ems that can be easily identified, while others
uire different experiments such as the NOESY
ent strategy used for the identification of the
acid for the GR1 protein was outlined by
region of the DQF-COSY spectrum by the
wo protons at the Cα formed two DQF-COSY
onents. Not all of the HN-Hα connectivities
ectrum for this work and therefore additional
ESY were used to aid in their assignment.
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
The methyl groups of alanine and threonine were found in the same area in the DQF-
COSY and TOCSY spectra. The methyl groups for both alanine and threonine were
found to be between 1-2 ppm and correlated to the Hα of alanine and the Hα & Hβ of
threonine at chemical shift values between 4-4.5 ppm.
The methyl resonances of valine Hγ, isoleucine Hγ2, isoleucine Hδ and leucine Hδ were
found within the same region as the methyl groups of alanine and threonine, but they
were well separated in the DQF-COSY and TOCSY spectra. The two-methyl
resonances of valine and leucine, with chemical shift values between 0.5-1.0 ppm
correlated to the Hβ for Val and Hγ for Leu with chemical shift values between 1.5-2.5
ppm. The methyl connectivities of Hγ2 and Hδ within isoleucine correlated with each
Connectivities were seen in the TOCSY spectrum between the Hα and 2 Hβ protons for
the amino acids serine, cysteine, tryptophan, phenylalanine, tyrosine, and aspartic acid.
The 2 Hβ peaks for the cysteine and aromatic amino acids were found between 2.8-3.6
ppm and for serine these peaks were located downfield between 3.5-4.0 ppm. The
confirmation of the aromatic and asparagine residues were determined by visualising
connectivities in the NOESY spectrum between the Hβ protons and the ring protons for
the aromatic residues or side chain amide protons for asparagine.
he remaining amino acids including lysine, arginine, glutamine, glutamic acid and
ied by their Hα-Hβ-Hγ-Hδ connectivities in the
other and occurred at chemical shift values between 0.5-1.0 ppm.
T
proline all contain similar Hα chemical shift values that were found to be between 4.3-
4.5ppm and each of these Hα protons correlated to a pair of Hβ protons that were found
between 1.7-2.2 ppm in the TOCSY spectrum. Proline residues do not contain a HN
chemical shift value and were identif
________________________________________________________________________________ 137
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
TOCSY/ NOESY spectra. Connectivities between the Hγ-Hδ of arginine and Hδ-Hε of
for the Hδ of Arg and
Hε of Lys and 1.7-2.1ppm for the Hγ of Arg and Hδ of Lys.
The Hα-HN regions of the DQF-COSY and TOCSY spectra were used to identify the
intra-residue spin systems of the amino acids of the protein. Figure 6.8 shows the
assignment of the residues found within the Hα-HN region of the TOCSY spectrum.
Not all of the peaks between 7.0 and 7.5 ppm in the TOCSY spectra were labelled in
Figure 6.10 as they corresponded to the side-chain N-H protons of arginine, lysine and
atic residues.
the GR1 protein.
lysine were found in the DQF-COSY spectrum at 3.1-3.6 ppm
arom
Figure 6.10: A summary of the identified spin systems in the TOCSY spectrum for
The spin systems were identified and labelled. The red lines were used for contrast in areas of
overlapping peaks. Three or more peaks were found under the red lines.
________________________________________________________________________________ 138
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
6.3.3 Sequential Assignment of the GR1 protein
Not all of the spin systems were identified using DQF-COSY and TOCSY experiments,
and additional experiments such as the NOESY, were used in the attempt to locate the
missing spin systems. The NOESY experiment was also used to sequentially assign
The XEASY program was used to analyse and document the peaks seen in the NOESY
spectrum (Bartels et. al., 1995). This program generates assignments and peak volume
tables that can be transferred into other programs such as DYANA, to create the three-
O
these residues by providing information on the intra-residues and inter-residue
connectivities within the protein.
Sequential assignment of the GR1 protein was obtained by looking for correlations
between adjacent spin systems, ie by viewing the Hαi-HNi+1, Hβi-HNi+1 and HNi-HNi+1
connectivities in the NOESY spectra as seen in Figure 6.11. The GR1 protein was
assigned sequentially until a proline residue (which has no amide proton) or 2 HN
resonances with similar chemical shift values (resulting in the overlapping of NOE
peaks within the spectra) were reached.
--- Ni-1 ---- CHi-1 ----- C ----- Ni ---- CHi ---- C ----- Ni+1 ---- CHi+1 ---- C ----
H H H
O O
CβHi-1 CβHi CβHi+1
Ri-1 Ri Ri+1
Figure 6.11: Sequential assignment of the protein.
________________________________________________________________________________ 139
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
dimensional structure for the protein. Table 6.1 summarises the assignment of the GR1
protein and Table 6.2 outlines the chemical shift values for the individual amino acids.
Ninety seven percent of the residues within the GR1 protein were assigned. The
remaining residues were not assigned due to regions of overlapping peaks in the DQF-
COSY, TOCSY and NOESY spectrums. This overlap made the definitive assignment
of the residues within the GR1 protein difficult and Figure 6.12 shows the extent of this
verlap in the TOCSY spectra.
Figure 6.12: The TOCSY spectra for the GR1 protein that shows the regions of
overlapping peaks.
ach boxed area corresponds to at least 3 different residues.
In attempts to overcome this problem, the experimental conditions were altered,
including reducing the temperature, adjusting the pH and varying the mixing times.
After reducing the temperature to 288K and adjusting the pH from 3.5 to 2.0, there was
very little difference within the overlapped regions. These overlapping peaks are also
o
E
________________________________________________________________________________ 140
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
apparent in the 13 C heteronuclear single-quantum correlation (HSQC) experiment,
especially within the region corresponding to the side-chain protons, but again very
little information could be obtained from this experiment (spectrum not shown).
The assignment of the GR1 protein was continued even though the overlapping peaks
were not assigned. The first residue was not assigned as no NOE connectivity was seen
between the Hα of residue 1 and HN of residue 2, and residues 18 & 31 could not be
assigned due to overlapping peaks.
C -Val-Cys and Cys-Arg-Cys, provided the starting point in
deciphering the NOESY spectra for the protein. The spin systems for the cysteine and
6.3.3.1 Sequential assignment of Residues 1-29
The first section of the GR1 protein to be assigned were residues 1 to 29 and the
connectivity plot is shown in Figure 6.13. Specific amino acid sequences within this
region, such as ys
valine residues were identified and the surrounding residues were sequentially assigned
using Hαi-HNi+1, Hβi-HNi+1 and HN-HN connectivities.
A break in the connectivity occurred as a result of two proline residues, found at
positions 18 and 19. The Hα protons of these proline residues were assigned from the
NOESY spectra. A strong NOE peak was seen between the Hα of Pro19 and HN of
Arg20 and NOE peaks were seen between the Hβ of Pro19 and HN of Arg20. Proline
18 was not completely assigned due to Hα chemical shift value for Pro18 being found
around the water peak (4.91ppm). Caution was taken with any peaks located in or
around the water peak, as it was unclear whether the peaks within this region were
________________________________________________________________________________ 141
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
artefacts. Peaks within this region may also be suppressed as a suppression sequence
was applied to the sample prior to acquiring data.
As a strong peak was detected between the Hα of Pro18 and Hδ of Pro19 in the
i i+1
Non-sequential connectivities were seen
Hα Pro19 & HN Ser16 suggesting these residues were involved in the
formation of a turn like structure.
This section of the GR1 protein contains 4 disulfide bridges that exist between Cys8-
Cys23, Cys11-Cys57, Cys13-Cys21 and Cys29-Cys34. Non-sequential NOE
connectivities between Hαi-Hβj of the cysteine residues identified the position of these
disulfide bonds. NOE connectivities were seen between Hα8-Hβ23, Hβ11- Hα57 and
Hα29-Hβ34. The identification of the Cys13-Cys21 disulfide bond was not
straightforward, as the Hβ protons of both cysteine residues were identical. However,
the presence of extremely large NOE peak volumes for the Hαi-Hβj and Hβi-Hβj
connectivities for these residues suggested the Hβ protons for both residues were
overlapped. Non-sequential connectivities were seen around the disulfide bridges at
HN14-HN20, Hα11-HN24, Hα11-HN23, Hα13-HN22, Hα21-HN14, HN12-HN22, HN14-Hβ20,
HN28-HN35 and Hα23-HN12.
NOESY spectrum and a peak was seen between the Hα Pro18 and Hα Ile17, the
assignment of the Hα of Proline 18 was confirmed. From this data, the cis/trans
conformation of the proline residues could be determined. Connectivities between the
Hαi-Hαi+1 of Ile17 and Pro18 showed this region to be in the cis conformation, while
the Hα -Hδ connectivities between Pro18 and Pro19 were consistent with the trans
conformation (Wüthrich et. al., 1984).
between the
________________________________________________________________________________ 142
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Figure 6.13: The fingerprint region of NOESY spectra of the GR1 protein at 303
K in 18% CD3CN/ H2O.
The one letter code is used to represent the amino acid residues. The blue and red lines indicate
the sequential connectivities between the residues 6-17 and Hα19-26 respectively.
The HN-HN connectivities seen in the NOESY spectra were used in conjunction with
the Hα-HN NOE peaks to provide sequential assignment of the residues within this
section by identifying neighbouring amino acid residues within the primary sequence.
Figure 6.14 shows the HN-HN connectivities for the residues in this section. There
were three non-sequential connectivities found within the HN-HN region - between
residues Thr14 and Arg20, between Val12 and Arg22 and between Val28 and Tyr35.
l HN-HN connectivities were also seen in this region of
These connectivities were located around the disulfide bridges of Cys13-Cys21 and
Cys29-Cys46. Non-sequentia
the Bowman-Birk inhibitor between Gln11-Ser25 and Thr15-Gln21 (Werner &
________________________________________________________________________________ 143
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Wemmer, 1991). These connectivities were also found near two disulfide bridges at
Cys14-Cys22 and Cys9-Cys24 in the Bowman-Birk inhibitor.
Figure 6.14: The HN-HN region of the NOESY spectra for the GR1 protein.
________________________________________________________________________________ 144
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
6.3.3.2 Sequential assignment of Residues 30-46
The second section to be assigned was for residues 30-46 and the HN-HN and the Hαi-
NHi+1 connectivities are shown in Figure 6.15. The identification of specific sequence
patterns and the remaining 2 valine spin systems within the protein allowed the
sequential assignment of this section. The HN-HN non-sequential connectivities within
the NOESY spectra aided in the assignment of a number of amino acid residues that
were located in overlapped regions (Figure 6.15A).
Non-sequential connectivities were detected between HN28-HN35, between Hα46-HN33,
between Hα20-HN45 and between Hα45-HN21. Disulfide bonds within this region were
located between residues Cys29-Cys34 and between Cys32-Cys46 and this was
confirmed by the presence of non-sequential connectivities between Hα29-Hβ34 and
between HN32-Hβ46.
The assignment of Pro39 was difficult, as no HN value could be determined for Ala40
in both the NOESY and TOCSY spectra. However, a strong NOE peak was seen
between Hα of Val38 and Hδ of Pro39, which allowed the proline residue to be
assigned. The second proline residue was assigned by identifying NOE peaks between
the Hα of Pro44 and HN of Tyr45 and between Hα of Arg43 and Hδ of Pro44. Both of
these proline residues were found to adopt the trans conformation as there were strong
sequential Hαi -Hδi+1 NOE cross peaks. This was a surprising find, as Pro39 was not
, the inhibitory studies completed on the GR1 protein
expected to be in the trans conformation due to its involvement in the inhibition of the
protease, chymotrypsin. However
did not show any reduction in the inhibitory effect of the protein towards chymotrypsin
________________________________________________________________________________ 145
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
due to the P3′ proline being in the trans-conformation. The KI values for the GR1
protein for chymotrypsin of 4.8 x 10-9M (Clague, 2001) are comparable with those
obtained for the Bowman-Birk inhibitor of 5.2 x 10-9M (Werner & Wemmer, 1991).
The assignment of the Hαi-HNi+1 NOE connectivities for this section was split into
three, due to the presence of two proline residues at positions 39 and 44. The
sequential assignments of these three regions are shown in Figure 6.15B. There were a
number of non-sequential medium ranged connectivities seen around Pro39 to Met42.
hese connectivities may be involved in forming a turn, similar to that seen for the first
T
region or trypsin binding site.
________________________________________________________________________________ 146
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
_
F
4
T
4
A
B
_______________________________________________________________________________ 147
igure 6.15: The (A) HNi –HN and (B) H tivities for residues 32-
thin the GR1 protein.
roline dues e the a ment of this region. Residues 32-38 are shown in green;
is in r 4-46 cyan. The one letter code was used to represent the amino acids.
i+1 α -HN connecI i+1
6 wi
wo p resi brok ssign
0-43 ed; 4 is in
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
6.3.3.3 Seque l assignment of Residues 47-61
ird a inal s on tha quentially assigned corresponded to the residues
. Th αi-N and H i+1 con f the remaining residues are
in F ure 6.1 This was r tforward to assign, as the
ity of rema g unas igned residu ere within this region. Specific amino
cids such s methi ine and soleucine we asily identified and assigned, as they
ad charac ristic peak patterns in the TOCSY spectra and only one was to be assigned.
position 58 was assigned by NOE connectivities between the Hα
nd Hδ of Pro58. The Hαi-HδI+1
on were assigned from the HN-HN and Hαi-HNI+1 regions of the
NOESY spectra. There were the occasional non-sequential connectivity between the
Hα53-HN57, Hβ53-HN57 and a disulfide bond was identified to exist between Cys11 and
Cys57.
ntia
The th nd f ecti t was se
48-61 e H Hi+1 Ni-NH nectivities o
shown ig 6. section elatively straigh
major the inin s es w
a a on i re e
h te
A proline residue at
of Pro58 and HN of Ile59 and between Hα of Cys57 a
connectivity resulted in Pro58 being in the trans conformation. A number of residues
within this regi
________________________________________________________________________________ 148
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
B
A
________________________________________________________________________________ 149
Figure 6.16: The (A) HN-HN and (B) HαI-HNi+1 connectivities for the residues 48-
61 seen in the NOESY spectra.
Residues 48-57 are shown in red and residues 59-61 are shown in blue. The one letter code for
the amino acids is used.
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
The summary of the sequential assignment for the amino acid residues within the GR1
protein are shown in Table 6.1 and the chemical shift values for these residues are
own in Table 6.2. Table 6.1 reveals a large number of sequential NOE connectivities
and very little medium range NOEs were seen for the GR1 protein. Interpreting the
ta seen within this table, it could be stated that there could be one region (residues 8-
27) that might contain β-sheet like structures due to the presence of a large number of
αN(i,i+1) and dβN(i,i+1) connectivities, while the second half of the GR1 protein looks
relatively disordered.
Table 6.1: The summary of the assignment of the GR1 protein.
sh
da
d
________________________________________________________________________________ 150
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Table 6.2: Chemical shift assignment of the GR1 protein in 18% CD3CN/ H2O pH 3.5 at 303 K.
Chemical Shifts in ppm Residue HN Hα Hβ Others
1. Gly ND ND 2. Gly 8.70 4.19 3. Glu 8.86 4.41 2.12 γCH2 2.46,2.24 4. Glu 8.50 4.17 2.09,2.02 γCH2 2.37
7. Trp 8.16 4.61 3.34,2.85#
9. Glu 7.73 4.12 2.29,2.17 γCH 2.47
11. Cys 7.75 5.54 3.42#,2.96
13. Cys 8.87 6.25 3.21 13 4.66 4.85 γCH3 1.63 55 4.75 2.27 γCH2 1.91; δCH2 3.42
16. Ser 7.57 4.61 4.15,4.01# 17. Ile 8.17 4.37 1.96 γ CH2 1.65,1.27;γCH3 1.07; δCH3 1.06 18. Pro -- 4.91 ND ND 19. Pro -- 4.40 2.61,2.06 γCH2 2.29,2.18; δCH2 3.70 20. Arg 7.63 5.58 2.04 γCH2 1.90; δCH2 3.44; εNH 7.46 21. Cys 8.58 5.99 3.21,2.96# 22. Arg 8.88 4.85 2.01 γCH2 1.81; δCH2 3.41; δNH 7.44 23. Cys 9.27 5.72 3.37,3.04 24. Thr 8.37 4.12 4.43 γCH3 1.50 25. Asp 8.16 5.08 3.23,2.74 26. Ser 8.83 4.55 4.20, 4.38 27. Ser 8.02 4.74 4.26 28. Val 7.55 4.44 2.66 γCH3 1.40, 1.28 29. Cys 8.22 4.55 3.35. 3.23 30. Thr 7.89 4.24 3.90 γ1.27 31. Lys ND ND ND ND 32. Cys 8.82 6.38 3.30,3.20 33. Val 9.26 4.58 2.24 γCH3 1.19 34. Cys 7.95 4.87 3.44,3.11# 35. Tyr 8.83 4.08 3.69, 3.56# δH 7.14, εH 6.87 36. Leu 8.23 4.33 2.02, 1.74# γCH2 2.39; δCH3 1.15 37. Thr 7.95 4.31 4.01 γCH3 1.34 38. Val 8.10 4.73 2.57 γCH3 1.40,1.01†
39. Pro -- 4.59 2.64 γCH2 2.24,2.01; δCH2 3.81, 4.05 40. Ala ND 3.91 1.62 41. Ala 8.79 4.34 1.64 42. Met 8.26 5.01 2.41,2.20# γCH2 2.89, 2.71 43. Arg 7.66 4.28 2.06 γCH2 1.88,1.57; δCH2 3.21 44. Pro -- 4.67 2.52 γCH2 2.13; δCH2 3.93, 3.55 45. Tyr 7.53 5.71 3.56,3.21 46. Cys 8.69 6.04 3.20,2.96 47. Glu ND ND ND 48. Ser 7.67 4.62 4.28#,4.10 49. Met 8.07 4.93 2.50#,2.40 δCH2 3.00,2.79 50. Ala 7.66 4.16 1.72
5. Ala 7.94 4.09 1.03 6. Asp 8.08 4.32 2.26, 2.08
δΗ 7.14;εNH 10.43; εH 7.59; ζH 7.71, 7.27; ηH 7.47 8. Cys 8.30 4.95 3.51,3.02#
2
10. Asp 8.98 5.10 3.37,2.88
12. Val 9.21 4.58 2.23 γCH 1.20,1.16
14. Thr 9.15. Arg 8.
3
________________________________________________________________________________ 151
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
Residue HN Hα Hβ Others 51. Ser 8.43 4.61 4.14 52. Arg 8.25 4.31 1.72 γCH2 1.59; δCH2 3.23; εNH 7.46 53. Phe 8.67 4.90 3.82,3.24# δH 7.42; εH 7.31; ζH 7.22 54. Asp 7.71 4.39 2.88,2.79# 55. Ala 8.63 4.20 1.12 56. Phe 8.03 4.56 3.60,3.28# δH 7.53; εH 7.46 57. Cys 7.54 5.75 3.57. 3.22 58. Pro -- 4.68 2.46, 2.15 γCH2 2.28; δCH2 4.12 59. Ile 8.46 4.44 2.10 γCH2 1.75,1.45; γCH3 1.17; δCH3 1.11 60. Gly 8.56 4.21 61. Ser 7.97 4.49 4.06, 4.21
ND = not determined # Indicates Hβ protons that were stereospecifically assigned † Indicates Hγ protons of Val that were stereospecifically assigned.
Referenced to an external sample of DSS at 0ppm that was prepared in 18% CH3CN-D3/H2O
pH 3.5 at 303K.
________________________________________________________________________________ 152
Chapter 6 NMR Assignment of the GR1 protein from Grevillea robusta ____________________________________________________________________________________
6.4 Conclusion
NMR spectroscopy was used to determine the structure of the GR1 protein. One-
dimensional and homonuclear two-dimensional NMR experiments were used to
identify the spin systems of the individual amino acids and sequentially assign these
residues within the GR1 protein. The spin systems of the amino acids were identified
by the DQF-COSY and TOCSY experiments, and the sequential assignment was
achieved by looking for Hαi-HNi+1 and HNi-HNi+1 connectivities in the NOESY
spectra. Ninety seven percent of the amino acid residues within the GR1 protein were
assigned. The remaining residues were not assigned due to a number of overlapping
peaks in the homonuclear 2D spectra. Connectivities between the Hαx-αHPro and Hαx-
HδPro in the NOESY spectra (where x is the preceding residue) determined the cis/trans
conformation of the proline residues. Five proline residues were found in the GR1
protein and all proline residues adopted the trans conformation with the exception of
Proline 18 where it was found in the cis conformation. There were very few medium
and long range NOE connectivities seen for the GR1 protein. However, the
connectivities that were seen did suggest one region may contain β-sheet like structures
based on the Hαi-HNi+1 and Hβi-HNi+1 connectivities.
________________________________________________________________________________ 153
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
Chapter 7 Structural studies of the GR1 protein
________________________________________________________________________________ 154
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
Chapter 7 Structural Studies of the GR1 protein
7.1 Introduction
This chapter outlines the strategy to predict the secondary structure of the GR1 protein
and the disulfide bond pattern for this protein. It also outlines the steps involved in
determining the final 3D structure of the GR1 protein and discusses the structural
differences between the final structure of the GR1 protein and the Bowman-Birk
inhibitor.
7.2 Secondary Structure of the GR1 protein
Prediction of secondary structure
The secondary structure of any protein can be determined by comparing the chemical
shift values for each amino acid with the random coil or theoretical chemical shift
he secondary structure for the protein can be predicted by reviewing the values
btained (random coil values – observed chemical shift values) and looking for stretches
7.2.1
values. A number of different methods were used to determine the random coil
chemical shifts, however, the shift values used in this work were derived from Wüthrich
(1986). The difference between the Hα values of the random coil chemical shift values
and the observed chemical shift values for the GR1 protein were calculated (Table 6.2
in Chapter 6) and graphed as seen in Figure 7.1.
T
o
________________________________________________________________________________ 155
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
of positive or negative values within this graph. Positive and negative values represent
regions of either α-helical or β-sheet like structures respectively. A continuous stretch
of 4 or more values of either negative or positive results strongly suggests the protein to
be in that conformation.
-2.00
1.00
0.50
-1.50
-1.00
-0.50
0.001 11 21 31 41 51 61
Residue number
d(rc
-obs
)
GR1 protein
Figure 7.1: Secondary structure prediction of the GR1 protein.
Wüthrich val
The values were determined by subtracting the observed Hα values from the protein with the
ues (Wüthrich, 1986). A negative result corresponds to the β-sheet conformation
and a positive value corresponds to the α-helix.
Regions of β-sheet like structures were predicted for residues 10-18, 20-23, 25-28, 44-
49 and 57-60 as they were found to contain stretches of 4 or more negative values as
seen in Figure 7.1. A number of regions contained a stretch of 3 consecutive negative
values and these regions may contain β-sheet like structures however, this can only be
final structure is determined. A disruption to the stretch of negative confirmed when the
values as seen for residues 18-20, may indicate the presence of a turn. Residues 3-6 and
________________________________________________________________________________ 156
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
54-56 contain a stretch of 3-4 consecutive positive values that could correspond to a α-
elical like structure. This structure may exist but can only be confirmed when the final
allel β-sheet structure, held together by a
etwork of disulfide bridges and hydrogen bonds (Werner & Wemmer, 1991; Werner &
man-Birk inhibitor.
h
structure of the GR1 protein is solved.
Secondary structure prediction was also applied to the Bowman-Birk inhibitor as this
structure was also determined by NMR spectroscopy (Werner & Wemmer 1991). As
there was significant sequence homology between the Bowman-Birk inhibitor and the
GR1 protein and the structure for the Bowman-Birk inhibitor was known, the two
sequences were aligned and the secondary structure prediction values were compared.
Figure 7.2 and Figure 7.3 show the comparison between the two proteins within the
trypsin- and chymotrypsin- binding regions of the Bowman-Birk inhibitor. The
numbering of the residues in the figures were based on the GR1 proteins’ sequence.
The trypsin-binding region showed both proteins to have a consecutive stretch of
negative values between residues 10-23 (Figure 7.2). Within the Bowman-Birk
inhibitor, this region was made up of an antipar
n
Wemmer, 1992). A disruption to the consecutive stretch of negative values as seen at
residue 19 was found to be characteristic of a turn, and this was confirmed by viewing
the structure for the Bowman-Birk inhibitor (Werner & Wemmer, 1992). Therefore,
reviewing the sequence alignment and the secondary structure prediction results, it
could be assumed that the trypsin-binding region of the GR1 protein would have very
similar structural characteristics with the Bow
________________________________________________________________________________ 157
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
________________________________________________________________________________ 158
: S o d comparison between the GR1 protein
(this work) and Bowman-Birk inhibitor (Werner & Wemmer, 1991) for the
trypsin-binding region
ues indicate a and
GR1 protein values are shown in
try i
e r s
not surprising to see a similar
-2
-1.5
-1
-0.5
1 16 10
0.5
1
1.5
1 6 1 2
Residue number
d(rc
-obs
)
BBIGR1
Figure 7.2 ec ndary structure pre iction
.
Negative val β-sheet conform tion α-helices are indicated by positive values.
The blue and the BBI values are shown in red.
The chymo psin-binding region of the Bowman-Birk inhib tor and the corresponding
region in th GR1 protein were compa ed and the difference are shown in Figure 7.3.
Residues 32-40 within the GR1 protein showed a number of sequence similarities with
the Bowman-Birk inhibitor, and therefore it was
functional behaviour between the two proteins. After residue 41 there was very little
sequence similarity, however, the secondary structure prediction between the two
proteins was similar. Stretches of negative values were identified for the GR1 protein
for residues 31-35 and 44-46, which suggested this region contained β-sheet-like
structures.
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
inhibitor (Werner & Wemmer, 1991) a
Figure 7.3: Secondary structure prediction comparison between the Bowman-Birk
nd the GR1 protein (this work) for the
hymotrypsin-binding region.
structure and the predicted α-helix like structures are represented by a positive value.
c
The GR1 protein is shown in blue and the BBI is in red. The negative values indicate a β-sheet
7.2.2 Secondary structure assignment
The sequential assignment revealed all proline residues were in the trans-conformation,
with the exception of Pro18, which was found in the cis-conformation, as there were
NOE connectivities between the HαIle17 and HαPro18 protons. The cis X-Pro peptide
bond was also seen in the Bowman-Birk inhibitor (Asn18-Pro19) and was involved in
forming the type VI turn within the trypsin-binding site (and also the chymotrypsin
binding site) of the inhibitor (Werner & Wemmer, 1992).
-2
-1.5
-1
0
0.5
1
-0.5
27 37 47
Residue number
d(rc
-obs
)
BBIGR1
________________________________________________________________________________ 159
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
All members of the Bowman-Birk family have been found to (a) contain a proline
residue at position P3′ and (b) have a cis- conformation between the P2′ and P3′ of the
anonical motif (McBride et. al, 1998). Several researchers have shown the highly
conserved proline residue in the P3′ position of the reactive site to be important for the
inhibition of the protease. They have also shown the level of inhibition was improved if
this proline residue was in the cis conformation rather than in trans (Brauer et. al.,
c
2002).
Hydrogen bonds were determined by detecting slowly exchanging amides. The sample
was dissolved in D2O at pH 3.5 at 288 K, and a series of 1D and TOCSY experiments
were completed over a period of 16 hours. Studying the amide protons within this
experiment provides information on the overall structure of the protein and reveals how
exposed these proteins are to the solvent (Wagner & Wüthrich, 1982). The slowly
exchanging amide hydrogens were Ala5, Cys11, Val12, Thr14, Ser16, Arg20, Arg22,
Thr37, Ser48 and Ser61. Hydrogen bonds may exist between Val12-Arg22 and Thr14-
Arg20 as NOESY connectivities were seen for these residues (shown by arrows in
Figure 7.4). Schematic representation of these potential hydrogen bonds is shown in
Figure 7.4.
Figure 7.4: Schematic representation of the hydrogen bonds between the residues
The arrows show NOE connectivities between residues. The dashed lines show possible
10-15 and 19-24.
hydrogen bonds.
________________________________________________________________________________ 160
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
7.3 Positioning of the disulfide bridges in the GR1 protein
The GR1 protein was characterised as a Bowman-Birk protease inhibitor based on the
sequence alignment studies (refer to section 3.6) and competitive inhibition work (refer
served cysteine residues as shown in the Figure
.5 represent the disulfide-bonding pattern within the Bowman-Birk inhibitors. Two
The GR1 protein contains 10 cysteine residues that form 5 disulfide bonds (determined
by mass spectroscopy; refer to section 3.7 for details). A number of these cysteine
residues align with those from the Bowman-Birk inhibitor family (Figure 7.5).
However the GR1 protein does not contain 4 of these cysteine residues. Therefore this
protein will mostly likely have a different disulfide pattern when compared to any of the
other members of the Bowman-Birk family.
to section 4.7). The Bowman-Birk inhibitor contains 14 cysteine residues
(approximately 20% of the total number of residues) that form 7 disulfide bridges, and
these cysteine residues are conserved within the Bowman-Birk family, suggesting they
play an important role in the function/ structure of these proteins (Gueven et al., 1998).
As all the cysteine residues within the Bowman-Birk inhibitor form disulfide bridges, it
can be assumed that these residues are important in maintaining the structure of the
inhibitor. Numbers underneath the con
7
disulfide bridges exist between the reactive sites for both trypsin (disulfide bond
numbers 2 and 4) and chymotrypsin (disulfide bond numbers 6 and 7) for the Bowman-
Birk inhibitors. The remaining 3 disulfide bridges are involved in maintaining the
overall structure of the inhibitor (Figure 7.5).
________________________________________________________________________________ 161
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
GR1 -- -- -- -- -- -- -- -- -- --
1 10 20 30
-- -- -- -- -- -- -- G G E E A D W C E D C V C
1BBI -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D D E S S K P C C D Q C A C 1PI2 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- D E Y S K P C C D L C M C IBB3_DOLAX -- -- -- -- -- -- -- -- D H H H S T D E P S E S S K P C C D E C A C IBB4_DOLAX -- -- -- -- -- -- -- -- -- H E H S S D E S S E S S K P C C D L C T C IBB_PHAAU -- -- -- -- -- -- -- -- -- -- -- -- S H D E P S E S S E P C C D S C D C IBB2_PHAAN -- -- -- -- -- -- S V H H Q D S S D E P S E S S H P C C D L C L C IBB3_SOYBN M C I L S F L K S D Q S S S Y D D D E Y S K P C C D L C M C IBB2_SOYBN -- -- -- M E L N L F K S D H S S S D D E S S D P C C D L C M C
1 2 3 4
31 40 50 60
GR1 T R S I P P R C R C T D S -- -- -- -- -- -- S V C T K C V C Y L T
1BBI T K S N P P Q C R C S D M R L N S C H S A C K S C I C A L S PI-II T R S M P P Q C S C E D R I -- N S C H S D C K S C M C T R S IBB3_DOLAX T K S I P P Q C R C T D V R L N S C H S A C S S C V C T F S IBB4_DOLAX T K S I P P Q C G C N D M R L N S C H S A C K S C I C A L S IBB_PHAAU T K S I P P E C H C A N I R L N S C H S A C K S C I C T R S IBB2_PHAAN T K S I P P Q C Q C A D I R L D S C H S A C K S C M C T R S IBB3_SOYBN T R S M P P Q C S C E D I R L N S C H S D C K S C M C T R S IBB2_SOYBN T A S M P P Q C H C A D I R L N S C H S A C D R C A C T R S
4 2 5 6 5 7
GR1 V P A A M R P Y
61 70 80 90
C E S M A S R F D A F C P I G S -- -- -- -- -- --
1BBI Y P A Q C F C V D I T D -- -- -- -- -- -- F C Y E P C K P S E D D PI-II Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB3_DOLAX I P A Q C V C V D M K D -- -- -- -- -- -- F C Y A P C K S S H D D IBB4_DOLAX E P A Q C F C V D T T D -- -- -- -- -- -- F C Y K S C H N N A E K IBB_PHAAU M P G K C R C L D T D D -- -- -- -- -- -- F C Y K P C E S M D K D IBB2_PHAAN M P G Q C R C L D T H D -- -- -- -- -- -- F C H K P C K S R D K D IBB3_SOYBN Q P G Q C R C L D T N D -- -- -- -- -- -- F C Y K P C K S R D D -- IBB2_SOYBN M P G Q C R C L D T T D -- -- -- -- -- -- F C Y K P C K S S D E D
7 6 3 1
Figure 7.5: The conservation of cysteine residues within a number of Bowman-
Conserved cysteine residues are boxed. The reactive sites for trypsin and chymotrypsin are
highlighted in yellow and red respectively. The number under each cysteine residue shows the
disulfide-bonding pattern for the Bowman-Birk inhibitor family. 1, GR1 (Grevillea robusta
Chen et. al., 1992). 4, 1BB3_DOLAX & 5, 1BB4_DOLAX (Dolichos axillaris inhibitor,
Jourbert et. al., 1979). 6, 1BB_PHA
Birk inhibitors and the GR1 protein (this work).
inhibitor). 2, 1BBI (Bowman-Birk Inhibitor, Werner & Wemmer, 1992). 3, PI-II Glycine max,
AU (Phaseolus aureus (mung bean) inhibitor, Zhang et. al.,
1982). 7, 1BB2_PHAAN (Phaseolus angularis (adzuki bean) inhibitor, Kiyohara et. al., 1981).
, 1BB3_SOYBN & 9, 1BB2_SOYBN (Glycine max, Joudrier et. al., 1987). 8
________________________________________________________________________________ 162
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
The disulfide-bonding pattern for the GR1 protein was identified by NMR spectroscopy
by looking for non-sequential NOE connectivities between Hαi-Hβj and Hβi-Hβj (Klaus
et. al., 1993). It was difficult to detect connectivities between the Hβi-Hβj protons, as
there was a significant number of other NOE peaks within this area. Connectivities
were however, seen for the following residues - Hα57-Hβ11, Hα29-Hβ34 and Hα8-Hβ23.
Due to both Hβ protons of Cys13 & Cys21 having identical chemical shifts, it was
difficult to assign this disulfide bond. However, the peak volumes for the Hαi-Hβj for
both residues were significantly larger than the other identified cysteine residues.
Therefore, the increase in peak volumes and the positioning of the cysteine residues
within the protein (Figure 7.5; disulfide number 4) confirmed the disulfide bond existed
between residues Cys13 and Cys21. As 4 of the 5 disulfide bridges were identified, the
final bridge existed between Cys32-Cys46.
The biochemical approach to the identification of the disulfide bonding patterns was
itations in finding the appropriate protease that
tion and identification of the cleaved fragments by
otential proteolytic cleavage sites, especially within the chymotrypsin region where the
considered however, there were lim
would cleave the GR1 protein. This approach requires the proteolytic cleavage of the
protein of interest, followed by isola
HPLC and N-terminal sequencing. The GR1 protein sequence was investigated for
p
positioning of the cysteine residues were not conserved. Ideally, the protein would need
to be cleaved between the Cys29 and Cys32 and between Cys34 and Cys46 to
determine the disulfide pattern within this region. However, there were very few
proteases that would cleave within this region due to the absence of key amino acids
such as aspartic acid, glutamic acid, lysine and arginine etc. This approach would not
provide enough data to determine the connectivities between the cysteine residues
________________________________________________________________________________ 163
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
within this region, and therefore, the biochemical approach to the identification of the
disulfide-bonding pattern for the GR1 protein was not used.
7.4 Three dimensional structure of the GR1 protein
7.4.1 S
Distance R1 protein were derived from the peaks assigned in the
NOESY with a mixing time of 200 ms. A number of different
mixing tim ent and, after insp ion of each of the
experime at the data acquired with a mixing time of 200ms
provided the best data to determine to structure of the GR1 protein despite the
possibility of artefacts being formed as the result of spin diffusion. Spin diffusion
occurs when there is indirect magnetisation of atoms from systems within the
vicinity o nte sity is related to the mixing time
and the d ixing time directly
tabulated
the XEASY software program. Peak volumes were defined by performing
n the NOESY spectrum. The peak
olumes were then used to generate distance restraints for calculating the structure of
tructural Restraints
restraints for the G
spectrum at 303 K
es were run for the NOESY experim ect
nts, it was decided th
other spin
f the NOE peak of interest. As the NOE i n
istance between the two interacting spins, the increased m
influences the intensity of the NOE peak and thus increases the chances of spin
diffusion occurring (Guntert 1998). All of the NOE peaks were recorded and
in
rectangular integration on each assigned peak i
v
the GR1 protein.
Stereospecific assignments were made by identifying peaks between HN-Hβ2, HN-Hβ3
and Hα-Hβ2 and Hα-Hβ3 on the β methylene protons and the γ-methyl groups of valine
________________________________________________________________________________ 164
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
in the NOESY spectrum. Seventeen stereospecific assignments were identified which
included one γ-methyl group of valine from the GR1 protein. Pseudoatoms were added
to the unassigned β-methylene and γ-methyl protons.
The structure predication program, dynamic algorithm for NMR applications or
DYANA (version 1.5; Güntert et. al., 1997) was used to generate the restraints from the
information generated in the XEASY program. A number of macros are written within
ino acid sequence, the
NOE peak volumes and assignment file th to create t e
s for the protein.
within DYANA was use to convert he NOE pea volumes into
r distance restraints. D on tiv os
s and the hydrogen bond w o A
distance restraints for the a ts
DYANA was used to generate the three dimensional structures of the GR1 protein. The
ANNEAL macro within DYANA uses the distance restraints generated by CALIBA to
create these structures. The ANNEAL macro was customised to calculate 100 structures
with a total of 10000 molecular dynamics steps followed by 1000 steps of minimisation.
The macro starts with 800 molecular dynamics steps at a temperature of 8.0 (Güntert et.
al., 1997). This was followed by a slow cooling stage over 9200 steps until the
temperature reached 0.0. After the molecular dynamics steps, 1000 steps of
DYANA that convert the information obtained from the NMR experiments into distance
and angle restraints. One such macro called CALIBA uses the am
s from e XEASY program h
DYANA restraint file
The CALIBA macro d t k
upper and lowe isulfide b ds connec ities, stere pecific
assignment value restraints ere added t the CALIB macro,
which calculated se addition l assignmen .
7.4.2 Structural Calculations
________________________________________________________________________________ 165
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
minimisation were applied to each structure. The temperature is a measure of the target
function units per degree of freedom.
7.4.2.1 Initial GR1 structures
The distance restraints for the structures generated for the GR1 proteins are shown in
Figure 7.6. Very few medium (R<5) and long-range (R>5) restraints were found
however these restraints were located throughout the whole protein suggesting the
protein had regions of well defined structure. The two regions corresponding to the
reactive sites of the protein were relatively exposed to the solvent and this was shown
by the rapid exchange of the Hα-HN protons (within the f 2hrs of the experiment).
Slowly exchanging amide protons were located surrounding the disulfide bridges
throughout the GR1 protein.
A number of preliminary DYANA structures were completed where only 10 structures
were generated to identify any major violations with the data. As there were a number
the peak volume value or by completely removing the assignment from the XEASY
database. Once all of the violating restraints were removed, the 100 initial structures
were generated with DYANA.
A total of 363 distance restraints were defined where 154 were intra-residue, 156 were
sequential, 18 were medium range and 36 were long range restraints (Figure 7.6). Due
irst
of overlapping peaks within the Hα-HN region of the NOESY spectrum, it was not
surprising to find distance restraint violations within the generated DYANA structures
as it was difficult to get an accurate peak volume value for some of the residues. The
violating distances were addressed by either reducing the rectangular integration box or
________________________________________________________________________________ 166
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
________________________________________________________________________________ 167
to a large number of overlapping peaks, the total number of distance restraints was
much less than expected for a protein of this size. The majority of the long- range
NOEs were located surrounding the disulfide bridges and the medium range NOEs were
found around regions that may correspond to turns.
Intra-residue connectivities are shown in white; sequential connectives are in light grey;
ROCHECK
was also used to determine the stereochemical quality of the generated structures and
these results, along with the RMSD values, are shown in the Table 7.1. For the 20
structures generated in DYANA with the lowest target function, 73.27% of their
residues were located in the most-favoured and additionally allowed regions, 21.64%
Figure 7.6: The number of NOE upper distance limits per residue in the amino
acid sequence of the GR1 protein.
medium range are in dark grey; and long-range restraints are in black.
The average global root mean square deviation (RMSD) values were determined for the
20 structures generated in DYANA with the lowest target function value, and these
values were determined for the three sections within the GR1 protein. P
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
were found in the generously allowed regions and 5.08% were located in the disallowed
gions. These results suggest the GR1 protein will need to be further refined before the
nal solution structure can be determined.
able 7.1: Summary of NMR restraints and structural statistics from DYANA for
ll 20 structures.
DYANA NOE distance restraints
re
fi
T
a
Total 363 Intra-residue 154 Sequential 156 Medium-range (R<5) 18 Long-range (R>5) 36 Max. NOE violation, Å 0.487 ± 0.08 Max. vdw violation, Å 0.232 ± 0.07 Residual Target Function 2.82 ± 0.48 Mean Global RMSD ‡ (Å)
4.20 ± 0.92 3.74 ± 0.90
3.51 ± 0.97 5.06 ± 1.11
Stereochemical (Ramachandran plot) quality #
Residues in disallowed regions (%) 5.08 ± 2.45
Res. 8-25 bb $ 2.85 ± 0.97 Res. 8-25 heavy Res. 30-48 bb $
Res. 30-48 heavy 5.37 ± 0.98 $Res. 48-61 bb
Res. 48-61 heavy
Residues in most/additionally allowed regions (%) 73.27 ± 5.17 Residues in generously allowed regions (%) 21.64 ± 4.27
‡ RMSD values were calculated using MOLMOL. $ bb refers to the backbone atoms N, Cα, C’.
7.5 Further refinement of the GR1 protein
The quality of the structures obtained from DYANA were assessed using the
minimisation program, SYBYL® program (Tripos Inc). Disulfide bonds and distance
restraints were added to each structure prior to minimisation to ensure these bonds were
constrained during the energy minimisation steps. A total of 8000 steps of conjugated
# Stereochemical quality was assessed using PROCHECK (Laskowski et. al, 1993).
________________________________________________________________________________ 168
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
gradient minimisation using the Tripos force field were applied to each of the 20
structures generated in DYANA with the lowest target function (Table 7.2). The
RMSD values were determined for the three sections within the GR1 protein and
PROCHECK was used again to determine the stereochemical quality of these refined
structures. The RMSD and PROCHECK results for the original DYANA structures and
SYBYL minimised structures are shown in Table 7.2.
Table 7.2: The summary of the 20 energy-minimised NMR structures of the GR1
DYANA SYBYL® minimisation
protein before and after SYBYL® minimisation.
R sidual Target Function 2.82 ± 0.48 -- -- -- SYBYL Total number of steps -- 2000 4000 8000 SYBYL Total energy (kcal/mol) -- 92.96 ± 17.13 76.41 ± 15.07 63.27 ± 15.60 Mean global RMSD‡ (Å) Res. 8-25 bb $ 2.85 ± 0.97 2.97 ± 0.96 2.98 ± 0.95 3.03 ± 0.93 Res. 8-25 heavy 4.20 ± 0.92 4.33 ± 0.99 4.34 ± 0.99 4.38 ± 1.00 Res. 27-48 bb $ 3.74 ± 0.90 4.11 ± 0.94 4.13 ± 0.94 4.18 ± 0.94 Res. 27-48 heavy 5.37 ± 0.98 5.58 ± 1.02 5.60 ± 1.01 5.63 + 1.00 Res. 50-61 bb $ 3.51 ± 0.97 3.05 ± 0.90 3.06 ± 0.90 3.37 ± 1.00 Res. 50-61 heavy 5.06 ± 1.11 4.58 ± 1.10 4.59 ± 1.09 5.05 ± 1.14 Stereochemical (Ramachandran Plot) quality # Residues in most/additionally allowed regions (%) 73.27 ± 5.17 82.34 ± 4.21 83.18 ± 4.33 83.46 ± 4.47 Residues in generously allowed regions (%) 21.64 ± 4.27 9.82 ± 3.31 8.64 ± 4.07 8.06 ± 3.78 Residues in disallowed regions (%) 5.08 ± 2.45 7.88 ± 3.36 8.16 ± 3.30 8.46 ± 3.08
e
‡ RMSD values were determined using MOLMOL. $ bb refers to the backbone atoms. #Stereochemical quality was assessed using PROCHECK.
The minimisation of the DYANA structures (with the lowest target function values) was
completed over three steps and these results are shown in Table 7.2. The total energy
calculated for the 20 structures decreased after each minimisation step suggesting the
structures had improved. This improvement was also seen in PROCHECK, where the
number of residues found in the most/ additionally allowed regions of the
Ramachandran plot had increased for all of the minimisation steps. There was a
________________________________________________________________________________ 169
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
significant reduction in the number of residues located in the generously allowed
regions (reduction of 13.5%) of the Ramachandran plot for the structures minimised
after 8000 steps. However, there was an increase in the number of residues found
within the disallowed regions from 3 to 4 residues after the final step of minimisation.
Overall, there was very little difference in the RMSD values obtained for the structures
generated before and after minimisation. After the first minimisation step, a slight
difference was seen but further minimisation steps did not result in any improvement to
these values. MOLMOL was used to visualise the structures generated before and after
minimisation and each section was individually studied.
The structures generated in DYANA and the SYBYL® minimised structures for
residues 8-25 are shown in Figure7.7. The RMSD values within this region were
consistent throughout each of the minimisation steps, however there was a slight
increase in these RMSD values when compared to the original DYANA structures, and
this increase was not apparent when the structures were visualised. The regions
surrounding the disulfide bridges overlay relatively well in both the DYANA and
SYBYL® structures. This was not unexpected as this region was heavily constrained
with distance restraints corresponding to the disulfide bridges and the non-sequential
NOE assignments. The loop region (residues 15-20) did not overlay very well
suggesting this region was more flexible within the GR1 protein.
________________________________________________________________________________ 170
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
___________________
Figure 7.7: Conve
are shown in red.
of constrained resid
of these residues
assignment of peak
residues surrounding
the minimised SYB
before and (B) afte
The disulfide bridges
There was very littl
GR1 protein (residu
The backbone for a
have a similar over
protein are again rel
Cys8
_________________________________________
rged structures of the residues 8-25
ues during the generation of these structur
were located within overlapping regio
volumes and therefore the generation o
the disulfide bond of Cys32-Cys46 were
YL® structures compared with the DYAN
r minimisation using SYBYL®.
formed across the region are shown in yellow
e change to the RMSD values obtained fo
e 29-48) for both the DYANA and SYB
ll 20 structures studied are shown in Fi
lapping pattern that suggests the residues
atively flexible. This flexibility could be
Cys8
A
BCys11
__________
of the GR
ue to the
es in DYA
ns, which
f distance
found to o
A structure
and the lo
r the seco
YL® gene
gure 7.8.
within th
d
Cys11
Cys13
Cys13 Cys21 Cys21Cys23
Cys23__________ 171
1 protein (A)
limited number
NA. A number
prevented the
restraints. The
verlay better in
s.
ng-range bridges
nd region of the
rated structures.
Both structures
is region of the
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
________________________________________________________________________________ 172
BA
steine residues that are involved in forming disulfide bonds are shown in red and yellow.
(A) 20 structures and (B) 10 structures shown.
The final region of the GR1 protein, residues 50-61, did not reveal any significant
differences between the two groups of structures generated by DYANA and SYBYL®
as seen in Figure 7.9. The RMSD values for both groups of structures revealed a slight
improvement for the minimised structures however this was not evident when the
structures were visualised.
Figure 7.8: Converged structures of the residues 29-48 of the GR1 protein (A)
before and (B) after minimisation using SYBYL®. The cy
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
Figure
before a
________
A
B7.9: Converged structures for the residues 50-61 of the GR1 protein (A)
nd (B) after minimisation using SYBYL®.
________________________________________________________________________ 173
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
7.6 The optimised 3D structure of the GR1 protein.
The SYBYL minimisation showed the original DYANA structures were of high quality,
this conclusion was based on the stereochemical quality of the structures, the
improvement in the total energy levels after each minimisation step and the consistent
RMSD values obtained throughout the minimisation process. Figure 7.9 shows the
solution s of the GR1 protein. The structura cteristics of each of the three
regions within the GR1 protein will be discussed in detail below. The structural
similarities and differences seen within these regions will be compared with those seen
w wman-Birk inhibitor.
Figure 7.10: The solution structure of the GR1 protein.
tructure l chara
ithin the Bo
The blue regions of the ribbon represent β-sheet like structures.
________________________________________________________________________________ 174
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
7.6.1 The structure of section one of the GR1 protein
Residues 8-26 of the GR1 protein were found to contain a short region of antiparallel β-
sheet structures held together by two-disulfide bonds at residues Cys8-Cys23 and
al disulfide bond was located
etween Cys11 and Cys57 and this bridge is thought to play a role in maintaining the
amide experiment (refer to section 7.2.2). The other amide hydrogens
identified from this experiment did not form hydrogen bonds.
Cys13-Cys21 (shown in Figure 7.11). An addition
b
structure of the GR1 protein. The antiparallel β-sheet region within the GR1 protein
was separated by a turn that was defined by identifying Hαi-HNi+3 and Hαi-Hδi+2 NOE
connectivities between Ser16 and Pro19 and between Thr17 and Pro19 respectively.
These patterns of NOE peaks were also seen for the Bowman-Birk inhibitor and are
known to form a type VI turn (Richardson, 1981; Werner & Wemmer, 1992). This
region also contains a cis X-Pro peptide bond at residues 18 and 19 and a trans X-Pro
between residues 17 and 18. An Hα-Hα NOE connectivity existed between the Ile17-
Pro18 residues which is characteristic of a cis-peptide. Hydrogen bonds were located
between HN12-O22 and HN14-O20 in the GR1 protein as seen in the slowly
exchanging
________________________________________________________________________________ 175
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
Figure 7.11: Converged structures of the trypsin-binding region of the GR1
protein.
Twenty structures with the lowest target function values were overlaid. The cysteine residues
rk
hibitor (Werner & Wemmer, 1991; Werner & Wemmer, 1992). These residues are
highlighted in blue within Figure 7.12. For example, an acid residue was found after a
in s o i s a g s o ted in
between two cysteine residues (position 6) and a basic residue was found in the P1
position of the reactive site. From these results, it was predicted that this region of the
-Birk inhibitor.
involved in forming disulfide bridges are shown in yellow and red.
Residues 8-26 of the GR1 protein were found to be sequentially similar with the trypsin-
binding region of the Bowman-Birk inhibitor as seen in Figure 7.12. There were
residues within the GR1 protein that were not conserved but contained similar
characteristics (basic, acid, non-polar, polar etc) with those found in the Bowman-Bi
in
cyste e (po iti n 3 within the f gure), a m ll non char ed residue wa l ca
GR1 protein would be structurally similar to the Bowman
________________________________________________________________________________ 176
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
GR1 7 W C E D C V C T R S I P P R C R C T D S -- 26BBI 8 C C D Q C A C T K S N P P Q C R C S D M R 28
Figure 7.12: The sequential alignment of the trypsin-binding region of the
The residues that are homologous are shown in red and the residues that are similar and could
substitute those seen in the Bowman-Birk inhibitor are shown in blue. The one letter code was
used for the amino acid residues.
Bowman-Birk inhibitor with the GR1 protein.
The Bowman-Birk inhibitor contains two distinct binding sites called the canonical
motif that is made up of 2 antiparallel β-sheet structures separated by a type VI turn
(Werner & Wemmer, 1991; Richardson, 1981). These antiparallel β-sheet and turn
regions were seen in the Bowman-Birk inhibitor and were also seen within these regions
of the GR1 protein as seen in Figure 7.13. NOE connectivities were seen between the
Hαi-HNi+3 and Hαi-Hαi+2 for the Bowman-Birk inhibitor, which defined the type of turn
(Richardson, 1981), and the formation of this turn was aided by the presence of a cis-
peptide between Ile19-Pro20. Within the GR1 protein, these NOE connectivities were
also seen, however the cis peptide was not located at the P3' position (at residue 18) but
instead the cis peptide was located at Pro19.
________________________________________________________________________________ 177
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
__
Fi
BA
th
h
is
RS
h
fr
T
T
lo
th
C
re
fo
(o
w
B
al
si
Arg15
__________________________________________
gure 7.13: Structural similarities betwe
e (B) Bowman-Birk inhibitor.
e cysteine residues that form disulfide bridges
boxed and shown in magenta and green. Th
CB database (PDB ID: 1BBI; Werner and Wem
e GR1 protein had three disulfide bonds w
cated between Cys8-Cys23, Cys11-Cys57
an-Birk inhibitor had four disulf
ys9-Cys24, Cys12-Cys58 and Cys14-Cys2
sidues form bridges across the β-sheet and
r P1-P1’ site) for the GR1 protein (Arg15
ithin the figure) were found to be in a simi
om the backbone and therefore, they are
e Bowm
r both proteins within Figure 7.13. The res
irk inhibitor. In order for these proteins to f
low adequate interaction with the protease,
de chain residues involved in interacting
Lys16
Ser16
__________
en the (A)
are shown
e Bowman-
mer, 1992
ithin the
and Cys1
ide bonds
2 (Figure
the locatio
dues invo
and Ser16
lar position
able to act
i
unction, th
trypsin (W
with the p
Ser17
Cys8
Cys23
Cys13
Cys21Cys11
Cys14
___________
GR1 after
in yellow and
Birk inhibito
).
trypsin-bind
3-Cys21 (F
located be
7.13B). Tw
n of these
lved in form
), (shown in
to those se
e residue
with nearb
es
erner & W
rotease wer
Cys22
Cys12
Cys24__
r
in
ig
tw
o
di
i
e
s
y
e
e
Cys9
___
mi
red
wa
g r
ure
ee
o
sul
ng
ma
n fo
mu
re
mm
po
Pro20
Pro19__________ 178
nimisation and
. The P1-P1’ site
s taken from the
egion that were
7.13A), while
n Cys8-Cys62,
f these cysteine
fides are shown
the reactive site
genta and green
r the Bowman-
st be exposed to
sidues from the
er, 1992). The
inting outwards
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
proteases. It was not surprising to see these residues in a similar position as the
ting trypsin by both proteins.
o loops that do not
ontain any obvious secondary structures such as α-helices or β-sheet. There was a fair
amount of flexibility around the backbone of the top 10 m d GR1 protein
ts between the residues within
f
the atoms have to move and the resulting structures are more likely to
inhibition assays revealed similar Ki values towards inhibi
7.6.2 The structure of section two of the GR1 protein
Residues 29-48 in the GR1 protein were found to contain disulfide bridges between
residues Cys32-Cys46 and Cys29-Cys34 (shown in yellow and red respectively within
Figure 7.14). These disulfide bonds resulted in the formation of tw
c
inimise
structures as shown in Figure 7.14. The presence of overlap could be due the decrease
in the number of medium and long-range distance restrain
this region. The more restraints added to the structures would limit the amount o
freedom
converge.
________________________________________________________________________________ 179
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
Figure 7.14: Top 10 refined structures for residues 28-48 in the GR1 protein.
bonds for residues Cys29-Cys34 and Cys32-Cys46 are shown in red and
.
cture of the chymotrypsin-binding site of the Bowman-Birk inhibitor was very
The disulfide yellow
respectively
The stru
different when compared to the GR1 protein. The sequence similarity between the two
proteins within the chymotrypsin-binding region was restricted to the reactive site
(shown in Figure 7.15 by the P1, P1’). Similarities existed between residues that
contained certain characteristics (acidic, non-polar etc) and these residues are
highlighted in Figure 7.15 along with the identical residues between both proteins.
GR1 27 -- S V C T K C V C Y L T V P A A M R P Y C E 47BBI 33 H S A C K S C I C A L S Y P A Q C F C V D I 54
Figure 7.15: The sequential similarities between the chymotrypsin-binding region
of the Bowman-Birk inhibitor and the GR1 protein.
Amino acids that were conserved between the two proteins are shown in red and residues that
could be substituted are highlighted in blue. Multicoloured lines show the disulfide-bonding
pattern within both proteins. The one letter code is used to represent the amino acids.
P1 P1’
________________________________________________________________________________ 180
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
The Bowman-Birk inhibitor contained three disulfide bridges between Cys32-Cys39,
ys36-Cys51 and Cys41-Cys49, which were involved in holding the β-sheet structures
gether, while the GR1 protein contains only two disulfide bridges between Cys29-
Cys34 and Cys32-Cys46. It is obvious from Figure 7.15, that these two proteins will
ave very different structure based on the positioning of the disulfide bridges. The
isulfide bonds within the Bowman-Birk inhibitor result in this region forming a “U”
haped structure that is highly restrained through these bonds. The GR1 protein on the
ther hand, will adopt an “S” like conformation that will have regions of flexibility due
the lack of the third disulfide found within the reactive site region.
The chymotrypsin-binding region of the Bowman-Birk inhibitor contained antiparallel
β-sheet structures and a type VI turn (Werner & Wemmer, 1991). The type VI turn
contained the NOE connectivities between the Hαi-HNi+3 between Ser44 and Ala47and
Hαi-Hαi+2 between Tyr45 and Ala47. A cis peptide bond was also seen between Tyr45
and Pro46 for the Bowman-Birk inhibitor and this aided the formation of the type VIb
turn. The chymotrypsin-binding region for the Bowman-Birk inhibitor is shown in
Figure 7.16A.
The GR1 protein does not contain any β-sheet like structures, instead it contains a
number of turns that are held together by two disulfide bridges to form two loops as
seen in Figure 7.16B. There are a number of small similarities between the two
proteins, however, the major difference is the presence of a disulfide bridge across this
region. The residues involved in forming the reactive site within the GR1 protein were
found to point away from the loop and therefore they are in the ideal position to act with
nearby residues from the protease as seen in Figure 7.15B.
C
to
h
d
s
o
to
________________________________________________________________________________ 181
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
The two proline residues found within this region of the GR1 protein were both in the
trans conformation, which differs from that seen for the Bowman-Birk inhibitor. The
proline at the P3′ position of the reactive site of the Bowman-Birk inhibitor (Pro46) was
in the cis-conformation which aids in the formation of the type VIb turn. As this proline
is conserved amongst all Bowman-Birk inhibitors including the GR1 protein, it must
play an important role in the function of the protein. The proline residue within the
GR1 protein at the P3′ position of the motif was found to be in the trans-conformation.
Therefore, as the GR1 protein was able to inhibit chymotrypsin at a similar level to that
seen for the Bowman-Birk inhibitor, it can be stated that the conformation of this
residue is not important for the functioning of the protein.
The residue numbers are shown for each protein. The disulfide bonds are labelled and shown in
Figure 7.16: The chymotrypsin-binding region of the (A) Bowman-Birk inhibitor
and (B) the minimised GR1 protein.
yellow and red. Proline residues are shown in cyan and the reactive sites within each protein are
boxed and shown in magenta and green.
A B
Cys39
Pro 46
Leu43
Ser44
Cys51Cys36
Cys41 Cys49
Leu 36Thr37
Pro39
Cys46Cys32
Cys34
Cys29
________________________________________________________________________________ 182
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
7.6.3 The structure of section three for the GR1 protein
The final section of the GR1 protein to be studied contained residues 50-61 which had
limited similarities with the Bowman-Birk inhibitor as seen in the Figure 7.17. There
are three residues that are identical between the two proteins, including a cysteine that
was found to form a disulfide bond with Cys11 in the GR1 protein, and the formation of
this bond was identical to that seen in the Bowman-Birk inhibitor.
GR1 48 S M A S R F D A F C P I G S 61
BBI 55 T -- -- -- -- -- D -- F C Y E P C 62
Figure 7.17: Sequence alignment of residues 50-61 from the GR1 protein with the
the Bowman-Birk inhibitor. milarities are shown in red for identical and blue for residues of similar characteristics.
or this region, the GR1 protein was defined as having a random coil like structure that
ndary structure prediction
gion
ight contain α-helical like structures however this was not the case, as seen in Figure
corresponding region inThe si
F
contained a bend between Arg52 and Phe56. The seco
completed using the chemical shift values for the GR1 protein indicated that this re
m
7.18.
________________________________________________________________________________ 183
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
__________________
The backbone is hig
The complete stru
in Figure 7.19. T
Figure 7.18: The
o distinct dom
structure and the r
inhibitor domain (L
tw
Ala50
_________________________________________
hlighted in blue.
ctures for the Bowman-Birk inhibitor and t
he Bowman-Birk inhibitor has a well orde
solution structure of the final section of t
ains while the GR1 protein consists of
emaining region held together by two disul
eu-Thr).
Ser61
_____________________ 184
he GR1 protein are shown
red structure that contains
he GR1 protein.
one region of secondary
fide bridges to provide the
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
S17
AFigure 7.19: Structural differences
(Werner & Wemmer, 1992) and (B) thBoth structures are shown in ribbon format
The two reactive sites are highlighted in mag
K16
L43
S44
S16
________________________________________
B
______________________ 185
between the (A) Bowman-Birk inhibitor
e GR1 protein (this work). where the disulfide bridges are shown in yellow.
enta and green.
R15
L36
T37
__________________
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
7.7 Conclusion
The structure of the GR1 protein was determined using data obtained from NMR
techniques and computer derived simulating annealing program (DYANA {Güntert et.
al., 1997}). Distance restraints were determined by using macros within DYANA and
additional restraints such as hydrogen bonds and disulfide bonds were added. Twenty
structures with the lowest target function values were chosen from the 100 structures
generated to be the initial solution structures of the GR1 protein. Further refinement of
the structures was achieved using minimisation with a conjugated gradient within the
SYBYL® software. The RMSD values and the stereochemical quality of the structures
were determined by using the MOLMOL and the PROCHECK programs respectively.
It was found that the structures had improved after the minimisation refinement with 43
residues in the most/allowed region (38 prior to minimisation). A decrease in the
number of residues were seen in the generously allowed region from 11 to 4 residues
and an increase was seen in the number of disallowed residues (from 3 to 4 residues).
As there was not a large increase in the number of disallowed residues, it was decided
that the solution structures for the GR1 protein, would be those that had undergone
minimisation using SYBYL®.
The solution structure of the GR1 protein is shown in Figure 7.20 and the residues for
both reactive sites within the protein are shown. Both of the residues within the reactive
sites were located at the top of the loop or turn-like structure, which provided easy
access to interact with their respective proteases. The sequence homology of the GR1
protein with the Bowman-Birk inhibitor provided some insight into the possible
structure of the protein. The trypsin binding region of the GR1 protein was very similar
to that seen in the Bowman-Birk inhibitor however, this was the limit of the similarity
________________________________________________________________________________ 186
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
______________________ 187
the GR1 protein was similar to that seen for the Bowman-Birk inhibitor and
erefore, the cis/trans-conformation at the P3' position of the reactive site was not
residues involved in inhibiting trypsin and chymotrypsin is shown in magenta and green.
chemical and structural data from the Bowman-Birk inhibitor
(Werner & Wemmer, 1991, 1992), provides a number of reasons why there were
differences in the two structures. Firstly, the Bowman-Birk inhibitor contained 2 extra
disulfide bonds when compared to the number of disulfide bonds the GR1 protein had.
Long range disulfide bridges throughout the Bowman-Birk inhibitor provided structural
support for the inhibitor. Secondly, the assignments of the NOE peaks for the Bowman-
Birk inhibitor (Werner & Wemmer, 1991) were more straightforward as this structure
did not have as many overlapping regions. As a result of this, there was a large number
between the two proteins. The proline residue at the P3' position in both the trypsin and
chymotrypsin binding regions for the GR1 protein was found in the trans-conformation
which was different to that seen for the Bowman-Birk inhibitor family. However, the
function of
th
important for the functioning of this protein.
Figure 7.20: The solution structure of the GR1 protein.
The backbone is represented by the ribbon structure. Disulfide bonds are in yellow and the
Reviewing the bio
R15
L36
S16
T37
__________________________________________________________
Chapter 7 Structural studies of the GR1 protein ____________________________________________________________________________________
of medium and long range NOE connectivities assigned for the Bowman-Birk inhibitor
and these assignmen s aid e e r o the solutio tu s providing
restraints for The ote had lar num o residues
located in overlapp regio s, w ic m ore
difficul e s
Leguminoseae m e
of the proteins within this fam ar e m n
first characterised y e lyc a t me b s of h s perfamily
have been isolated from g e n ; h n et al., 1 8 , Winter pea
al., 1981) to name a few. The GR1 protein is the first member of the
Proteaceae family to be characterised as a Bowman-Birk inhibitor.
t ed in th g ne ation f n struc re by
all of the residues. GR1 pr in a ge ber f
ing n h h ade the assignment of NOE peaks m
t. Finally, most of the m mber of the Bowman-Birk superfamily belong to the
fa ily and it could therefore be assum d that the function and structure
ily would be simil . Th Bow a -Birk inhibitor was
from so b ans (G ine m x) and o her m er t is u
mun b a (Phaseolus aureus Z a g . 9 2)
(Pisum sativum; De La Sierra et. al., 1999), Adzuki bean (Phaseolus angularis;
Kiyohara et.
________________________________________________________________________________ 188
Chapter 8 Conclusions ____________________________________________________________________________________
Chapter 8 Conclusions
________________________________________________________________________________ 189
Chapter 8 Conclusions ____________________________________________________________________________________
Chapter 8 Conclusions
Two functional proteins were isolated from the seeds of Grevillea robusta: a lectin
(GR2; Chapter 5) and a serine protease inhibitor (GR1; Chapters 3 & 4). The lectin
targeted specifically white blood cell receptors and therefore could be used to
characterise the white blood cell receptors (Chapter 1). The serine protease inhibitor
was found to inactivate both trypsin and chymotrypsin independently and has potential
application in the generation of insect resistant plants or in the treatment of specific
cancers (Kennedy, 1998a; Chapter 4). These proteins were isolated and characterised
by a number of different techniques including gel electrophoresis, affinity, ion exchange
and gel filtration chromatography, mass spectroscopy, N-terminal sequencing and
specific bioassays. The extract was shown to have lectin properties and the protein was
present in low yields within the seeds of G.robusta (Chapter 2). The lectin was
Agglutination, binding and sugar specificity confirmed the isolated protein was a lectin,
which was mannose specific. The lectin isolated in G.robusta was called the GR2
protein.
A serine protease inhibitor was isolated by two different methods: (1) ion exchange &
RP-HPLC and (2) size exclusion chromatography. A number of different conditions
such as the salt concentration, the pH and the gradient applied to the column were
applied to the ion exchange column, to ensure the maximum separation of the proteins
in the crude extract. Two methods were derived due to the presence of a contaminating
protein seen on native PAGE, which was later identified by N-terminal sequencing as a
cleaved product of the GR1 protein. Therefore, to minimise the presence of
contaminating proteins, a second method was developed which involved the addition of
successfully isolated with affinity chromatography using an oligosaccharide matrix.
________________________________________________________________________________ 190
Chapter 8 Conclusions ____________________________________________________________________________________ protease inhibitors to the extraction buffers. The resulting protein was 95% pure
etermine by visualisation on native PAGE. Mass spectroscopy confirmed the proteins
isolated from the two different methods were identical and had a molecular weight of
6669Da.
he full amino acid sequence of the GR1 protein was determined using cDNA
techniques (Clague, 1999). The GR1 protein was found to have significant sequence
omology with a number of known serine protease inhibitors, especially those within
the Bowman-Birk superfamily. Within the Bowman-Birk family, the cysteine residues
nd those involved in forming the reactive site are conserved, which suggested these
ites played an important role in the structure/function within the protein. All of the
ysteine residues within the Bowman-Birk inhibitor are involved in forming disulfide
e GR1 protein, a number of
cysteine residues and other key residues were found in the same position as those seen
r the Bowman-Birk inhibitors. However, the total number of cysteine residues within
e GR1 protein was less than that seen for the Bowman-Birk inhibitors and this was
evident in the chymotrypsin-binding region, resulting in a different disulfide-bonding
pattern for the GR1 protein. Figure 8.1 outlines the similarities and differences between
the two proteins.
d
T
h
a
s
c
bridges, which provide structure to the protein. Within th
fo
th
________________________________________________________________________________ 191
Chapter 8 Conclusions ____________________________________________________________________________________
1 10 P1 P1’ 20
GR1 -- G G E E A D W C E D C V C T R S I P P BBI D D E S S K P C C D Q C A C T K S N P P 21 30 40
GR1 R C R C T D -- -- -- -- S -- -- S V C T K C V BBI Q C R C S D M R L N S C H S A C K S C I 41 P1 P1’ 50 60
S M A S R F D GRI C Y L T V P A M R P Y C E BBI C A L S Y P A Q C F C V D I T D -- -- -- -- 61 70
GR1 A F C P I G S -- -- -- -- -- -- -- -- -- -- BBI -- F C Y E P C K P S E D D K E N --
Figure 8.1: Sequence alignment of the GR1 protein and the Bowman-Birk
The residues that are conserved between the two proteins are shown in red. The cysteine
residues are shown in dark blue and those that are conserved are boxed. The scissle bonds for
the two inhibitory sites within the Bowman-Birk inhibitor (BBI) are shown in blue.
inhibitor.
Competitive inhibition assays were completed and confirmed the initial findings of the
GR1 protein belonging to the Bowman-Birk superfamily of serine protease inhibitors.
The GR1 protein inhibited both trypsin and chymotrypsin with Ki values comparable to
those seen for the Bowman-Birk inhibitor.
NMR spectroscopy was used to determine the three-dimensional structure of the GR1
protein. Homonuclear experiments two-dimensional experiments were used to identify
the individual spin systems for each amino acid and was also used to sequentially assign
the residues in the GR1 protein. Ninety seven percent of the residues were assigned
within the protein and the remaining 3% were not assigned due to a number of
overlapping peaks within the 2D spectrum. The cis/trans conformations of each of the
5 proline residues were determined by looking for Hαi-Hαi+1 and Hαi-Hδi+1 NOE
________________________________________________________________________________ 192
Chapter 8 Conclusions ____________________________________________________________________________________ connectivities. Four of these residues adopted the trans conformation and Proline 18
as in the cis conformation.
The initial GR1 structures were determined by using the computer program DYANA
(version 1.5; Güntert 1997). Distance restraints, stereospecific assignments and
hydrogen bonds were calculated using the CALIBA macro within DYANA. One
hundred structures were generated using the ANNEAL macro where each structure
underwent 10000 steps of molecular dynamics followed by 1000 steps of minimisation.
Twenty of the generated GR1 structures with the lowest target function values
nderwent further refinement using the SYBYL® program. A total of 8000 steps of
conjugated gradient minimisation steps were applied over 3 stages to the DYANA
structures. The stereochemical quality of the minimised structures were determined by
ent in the total number of residues in the
on were reduced (from 11 to 4 residues)
region increase from 3 to 4 residues. As
r ructures had improved from the original
DYANA structures, the solution structures for the GR1 protein were defined as those
w
u
PROCHECK and resulted in an improvem
most/allowed region of the Ramachandran plot (from 38 to 43). The total number of
residues found in the generously allowed regi
and the number of residues in the disallowed
the ste eochemical quality of the minimised st
generated after minimisation using SYBYL®.
________________________________________________________________________________ 193
Chapter 8 Conclusions ____________________________________________________________________________________
antiparallel β-sheet structures that
ere separated by a turn. The turn in the Bowman-Birk inhibitor was defined by a cis-
peptide between Asn18-Pro19. However, the cis-peptide within the GR1 protein was
located between the Pro18-Pro19 residues and therefore, the type of turn seen in the
GR1 protein was different from the Bowman-Birk inhibitor. The chymotrypsin-binding
region of the GR1 protein was very different from the Bowman-Birk inhibitor. This
region was made up of a number of turns and loops that were held together by 2
disulfide bridges. Sequence similarities existed for the key residues within the reactive
site, which explained the similar functional properties of the GR1 protein.
The GR1 protein contained one region of secondary structure that was made up of an
antiparallel β-sheet-like structure, which corresponded to the trypsin-binding region.
A
There were a number of structural similarities and differences between the GR1 protein
and the Bowman-Birk inhibitor. The trypsin-binding regions within both proteins were
sequentially and structurally similar with each other as seen in the previous chapter
(Figure 7.18). Both proteins contained regions of
w
A
se
disulfide bridges (Cys29-Cys34 & Cys32-Cys4
the Bowman-Birk inhibitor.
The cond region of the protein exposed l
the GR1 protein that were responsible for in
regions that could be easily accessible by t
conserved in both reactive sites (ie in the P3'
conformation rather than the cis-conformatio
within this family. It was thought that the cis
within the protein (McBride et. al., 1998), how
protein as the KI values obtained for this prot
_____________________________________________
B
B
_______ 194
6) to fold the protein. Both sites within
eucine 36 and threonine 37 using two
hibiting the proteases, were located in
he protease. The two proline residues
position), were found to adopt the trans-
n seen in all other protease inhibitors
-peptide improved the level of inhibition
ever, this was not the case for the GR1
ein were comparable with those seen for
____________________________
Chapter 9 Experimental _____________________________________________________________________________________
___________________________________________________________________ 195
Chapter 9 Experimental
Chapter 9 Experimental _____________________________________________________________________________________
Chapter 9 Experimental
9.1 Extraction of proteins from ground seed material.
9.1.1 Ammonium sulfate precipitation of proteins.
The crude protein sample was extracted from the seeds of G.robusta using a two-step
ammonium sulfate precipitation. Seeds were ground in a commercial grinder and
oaked in PBS at pH 7.3 (11.3 mM disodium hydrogen orthophosphate (BDH), 1.28
o hosphate (BDH), 140 mM sodium chloride (Sigma))
overnight at 4ºC slowly stirring. Ground material was removed by filtering through
cheesecloth followed by centrifugation at 48400x g for 30 minutes at 4ºC. Ammonium
sulfate (Sigma) was added to the supernatant to create a 0-40% saturation range (22.6
g/100 ml of supernatant) and incubated for a further 6 hours at 4ºC. Centrifugation
removed the precipitated material (unwanted proteins) and a 40 –80% saturation range
was created using ammonium sulfate to precipitate the proteins of interest. Precipitated
material was removed by centrifugation (48400 x g, 30 minutes at 4ºC) and the pellet
was resuspended in milli-Q water. Dialysis was used to remove excess ammonium
te ing against milli-Q water (30 minute
hanges for 3 hours) followed by dialysing against PBS pH 7.3 (30 minute changes for
4 hours). Due to the majority of proteins containing a molecular weight of 14000 Da or
below, dialysis tubing with a MWCO of 7000 Da was used (Selby Biolab).
s
mM s dium dihydrogen orthop
sulfa from the “crude” proteins by initially dialys
c
___________________________________________________________________ 196
Chapter 9 Experimental _____________________________________________________________________________________
9.1.2 Ammonium sulfate precipitation of proteins using protease inhibitors.
he proteins were extracted from the seeds of G.robusta using protease inhibitors in the
extraction buffer containing a mixture of protease inhibitors (TBS pH 7.8 (20 mM
Trizma (Sigma Aldrich), 0.15 M sodium chloride), protease inhibitor cocktail (PIC) (1.5
ml; Sigma Aldrich) and 0.25 mM PMSF (Sigma Aldrich) where they were stirred
overnight at 4ºC. The initial extraction of the crude extract followed the same method
as that outlined in section 10.1.1. Dialysis of the precipitated material was initially
against milli-Q water followed by extraction buffer containing protease inhibitors.
9.2 Polyacrylamide gel electrophoresis
The Mini Protean 3 electrophoresis equipment from Bio-Rad Laboratories was used to
prepare and run the polyacrylamide gels.
9.2.1 Sodium Dodecyl Sulfate (SDS) PAGE
An 18% separating gel with a 5% stacking gel was prepared following the method
developed by Laemmli (1970). The protein sample (20 µg) was diluted 1:2 with
reducing sample buffer (0.06M Tris-HCl, 3.2% glycerol (BDH), 2% SDS, 5% β-
mercaptoethanol (Bio-Rad laboratories) and 0.01% bromophenol blue) and heated to
100°C for 5 minutes to ensure the protein was in its reduced state. The protein was
loaded onto the gel with LMW standards (Pharmacia Biotech) and the gel was run at
constant voltage at 100 V until the dye front reached the bottom of the glass plates. The
T
extraction buffer to prevent proteolysis. Seeds were ground as before and soaked in
___________________________________________________________________ 197
Chapter 9 Experimental _____________________________________________________________________________________
LMW markers used were phosphorylase B (Mr 96 kDa), bovine serum albumin (67
kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa)
and α-lactalbumin (14.4 kDa). The gels were stained with Coomassie Brilliant Blue
(0.1% Coomassie blue R-250 in 10% acetic acid, 30% methanol in water) and destained
using 10% acetic acid and 30% methanol in water.
9.2.2 Native PAGE
Native polyacrylamide gels were prepared as outlined by Ornstein (1964) and Davis
(1964). A 13% separating gel with a 3% stacking gel was made. Samples containing
40 µg of protein were diluted 1:4 with 4 x native sample buffer (1.98 M Tris-HCl, 4%
glycerol and 0.01% bromophenol blue). The gel was run at a constant voltage of 100 V
initially and then increased to 200 V after the sample had migrated into the separating
gel. Coomassie Brilliant Blue was used to stain the gels.
9.3 Protein concentration estimation
A known protein standard was used to create a standard curve for the assay. Bovine
serum albumin (1 mg/ml)(BSA; Sigma Aldrich) was diluted to form a range of different
concentrations from 5 – 160 µg/ml. Using a 96 well microtitre plate, 100 µl of standard
(at each concentration) and 100 µl of protein sample was added to the wells (in
triplicate). To these wells, 100 µl of Bradford’s reagent was added (Sigma Aldrich)
and the plate was mixed carefully. A plate reader was used to determine the absorbance
of the samples at a wavelength of 595 nm. The results obtained for the standards were
___________________________________________________________________ 198
Chapter 9 Experimental _____________________________________________________________________________________
graphed against its respective concentration to provide a standard curve. This curve
allowed the concentration of the protein samples to be determined.
9.4 Purification of the GR1 protein - Part 1
The BioLogic HR chromatography system was used to monitor the ion exchange and
gel filtration columns. It contained an in-line conductivity and UV monitor that allowed
The Q sepharose FF column (25 x 200mm) (Amersham Pharmacia Biotech) was
equilibrated with the 20 mM Tris-HCl pH 8.5 at a flow rate of 4 ml/min. The step
gradient 0-12%, 12-23%, 23-35%, 35-100% 0.5M NaCl/ 20 mM Tris-HCl pH 8.5 was
used to separate these proteins. The proteins were detected using UV at 280 nm.
Fractions were pooled and freeze dried. Once dried, these samples were resuspended in
PBS pH 7.3 and dialyzed against this buffer to remove excess salt.
the detection of proteins and ionic strength to be recorded. The Waters HPLC system
was used to elute proteins from a reverse phase HPLC column. It contained an auto-
injection carousel and UV was used to detect eluting proteins.
A number of difference methods were applied in order to separate the proteins from
G.robusta. The methods listed below are those used to produce the final product.
9.4.1 Ion exchange chromatography (IEX)
___________________________________________________________________ 199
Chapter 9 Experimental _____________________________________________________________________________________
9.4.2 Reverse phase-HPLC
The SO2 protein eluted from ion exchange chromatography was further purified using
reverse phase HPLC. A C-18 column (Rainin; 10 x 50mm) was used and equilibrated
with 0.1% TFA in water. Acetonitrile was used to elute the proteins from the column
by applying a continuous gradient (25-32%). A second method was developed to
decrease the time between runs. An isocratic flow rate at 29% acetonitrile/ 71% TFA in
water (0.1%) was used and the eluted peaks from both methods were pooled and freeze
dried.
9.4.3 High Q chromatography
A High Q column (10ml; Bio-Rad laboratories) was prepared by equilibrating it with 20
mM Tris pH 8.5. The fraction containing the SO2/4 proteins (Qseph.8.5.Pk7) was
9.5.1 Gel filtration chromatography
Proteins were isolated using a superdex 75 column (Amersham Pharmacia Biotech) (25
x 200mm) that was equilibrated with TBS + PI pH 7.8. The crude extract prepared with
protease inhibitors was injected onto this column and the proteins were eluted using the
injected onto this column and washed with equilibrating buffer. A continuous gradient
(0-30% 0.5 M NaCl/ 20 mM Tris pH 8.5) was applied over 30 minutes at a flow rate of
2 ml/min. Fractions were pooled and freeze dried.
9.5 Purification of the GR1 protein – Part 2
___________________________________________________________________ 200
Chapter 9 Experimental _____________________________________________________________________________________
equilibration buffer at a flow rate of 2 ml/min. Eluted proteins were pooled and
concentrated by freeze-drying.
9.6 Purification of a lectin from G.robusta
The lectin from G.robusta was purified using affinity chromatography. A mannan-
agarose column (10 ml) was equilibrated with PBS pH 7.3 and the crude extract was
applied at a flow rate of 1ml/min. The column was washed three times with PBS pH
7.3 and the lectin was eluted using 0.5 M mannose in PBS pH 7.3. The fractions
ontaining the lectin were pooled and dialyzed initially against water for two 30 minutes
e pH 7.3 for a further hour. The lectin was concentrated by
freeze-drying.
9.7.1 Deglycosylation of proteins
ase II (10U/ml in 20 mM Tris-HCl pH 7.5, 25 mM NaCl) and 2 µl of O-
lycosidase (1 U/ml in 20 mM Tris-HCl pH 7.5, 25 mM NaCl) was added to the protein
and incubated at 37°C for 1 hour. Ten µl of water, 10 µl of pH adjustment buffer (0.5
M sodium phosphate dibasic) and 2 µl of PNGase F (2.5 U/ml in 20 mM Tris-HCl pH
c
chang s and then against PBS
9.7 N-terminal sequencing of proteins isolated from G.robusta
An enzymatic deglycosylation kit was purchased from Bio-Rad laboratories and used to
prepare the proteins. Twelve µl of sample (containing up to 100 µg of protein) was
diluted with 4 µl of 5x reaction buffer (250 mM sodium phosphate pH 6.0). A further 2
µl of NAN
g
___________________________________________________________________ 201
Chapter 9 Experimental _____________________________________________________________________________________
7.5, 50 mM NaCl, 1 mM EDTA) was added to the reaction vial. The sample was
incubated for a further 24 hours at 37°C. Samples were stored at 4°C until needed.
stem was packed in ice to provide better results. The
embrane was stained with Coomassie Blue in 50% methanol and destained with 50%
methanol. The bands were removed from the membrane and sent to the Protein centre
at the University of Queensland Biochemistry department to be sequenced for a fee.
9.8 Bioassays
9.7.2 Native PAGE and Electroblotting of proteins
A 13% native separating gel with a 3% stacking gel was prepared for sequencing. The
deglycosylated proteins were loaded onto the gel and run at 150 V until the dye front
was at the bottom of the glass plates. The proteins were electroblotted onto Sequi-blot
PVDF membrane (Bio-Rad laboratories) at 100 V for 90 minutes using the Bio-Rad
mini Trans blot system. The buffer used to transfer the proteins onto the membrane
(Towbin buffer; 25 mM Tris, 192 mM glycine, 20% methanol) was cooled to 4°C prior
to use and the Trans-Blot sy
m
9.8.1 Biotinylation of proteins
Samples to be tested in the GIFT were biotinylated prior to test being set-up. The
samples were dialyzed against 0.1 M sodium bicarbonate pH 8.5 for 1 ½ hours (two
changes of buffer at 45 minutes each) at room temperature using dialysis tubing with a
MWCO of 6000-8000 Da (Selby). The volume was measured for each sample and 16
µl of biotin solution (0.02 g in 1ml of DMSO) per 100 µl of sample was added to the
___________________________________________________________________ 202
Chapter 9 Experimental _____________________________________________________________________________________
protein. The sample was covered in foil and incubated at room temperature for 2 hours
rocking slowly. The excess biotin was removed by dialyzing against PBS pH 7.3 for 1-
½ hours at room temperature (two changes every 45 minutes). The sample was stored
9.8.2 Granulocyte harvest
Anti-coagulated blood was collected in 10 ml EDTA tubes and centrifuged at room
temperature for 10 minutes at 100 g. The platelet rich plasma (PRP) was removed and 2
m chloride) and 1 ml of Bornes EDTA/BSA (18 mM
orthophosphate, 9 mM sodium-EDTA, 0.15 M sodium chloride, 0.8 mM
d at room temperature for 15 minutes at 1600 x g. Granulocytes were
arvested from the middle layer of the double gradient and washed in Bornes
at -20°C.
mls of 5% Dextran (in 0.9% sodiu
disodium
sodium azide and 0.2% BSA) was added to each of the 10 ml tubes. The tubes were
mixed by inversion and incubated at a 45-degree angle for approximately 30 minutes at
37°C. The leukocyte rich plasma (LRP) was removed and transferred into graduated
plastic conical tubes (4 mls maximum volume per tube). A double density gradient was
formed using 9” glass pipettes and by placing 2.5 ml of Ficoll Paque (density of 1.077)
followed by 2.5 ml of Mono-Poly resolving medium (density of 1.114). The tubes were
centrifuge
h
EDTA/BSA three times. After the final wash, the cells were resuspended in 1ml of
Bornes EDTA/BSA and counted on a Sysmex K-100. Cells were then diluted to the
required concentration for each of the tests.
___________________________________________________________________ 203
Chapter 9 Experimental _____________________________________________________________________________________
9.8.3 Granulocyte Agglutination Test (GAT)
After counting the cells, the granulocytes were diluted to a final concentration of 5 x
6 l tion (GRS; 20 mls of Bornes EDTA/BSA,
0.1 g of EDTA & 260 µl of 30% BSA). Paraffin oil was placed into the wells of a
s read on an inverted phase
icroscope and graded depending on the strength of reaction (0-4+).
After the cells were counted, half of the cells were removed for other tests (eg the GAT)
and the remainder was centrifuged in an immufuge for 2 minutes on ‘HIGH’. The cell
button was resuspended in 1 ml of 1% paraformaldehyde and incubated for 2 minutes at
room temperature to fix the cells. The granulocytes were washed with Bornes
EDTA/BSA twice and diluted to a final concentration of 10 x 10 /ml.
Twenty-five µl of cells (at 10 x 106/ml) were pipetted into the wells of a V-bottom
microtitre plate. For control samples, 50 µl was added to the wells. The plate was
centrifuged at 3000 rpm for 2 minutes and the buffer was removed from the cell pellet
by flicking and draining onto a tissue. Each cell pellet was resuspended in 30 µl of
as
10 / m with granulocyte resuspension solu
microtest culture plate and 3 µl of test sample was placed in duplicate under this oil in
the middle of each well. One µl of cell suspension was added to the sample in each well
using a multi-dispense syringe (dispensing in 1 µl aliquot). Between samples, 3 µl of
cells were expelled and the needle wipes to prevent contamination of the samples. The
trays were incubated at 30°C for 3-4 hours. The plate wa
m
9.8.4 Granulocyte Immunofluorescence Test (GIFT)
6
biotinylated testing sample and 60 µl was added for the control samples. The plate w
___________________________________________________________________ 204
Chapter 9 Experimental _____________________________________________________________________________________
sealed and incubated at 30°C for 30 minutes. Unbound material was removed by
washing the plate twice with Bornes EDTA/BSA and centrifuging at 3000 rpm for 2
minutes. The cell pellet was resuspended in 30 µl of streptavidin-FITC (1:45 dilution in
Bornes EDTA/BSA) in each well (60 µl for control samples), sealed and incubated at
The samples to be tested were biotinylated as outlined in section 10.8.1. Different
concentrations were made up of the 8 chosen sugars (0.1 M, 0.25 M & 0.5 M). The
biotinylated sample to be tested for sugar specificity was diluted in a 1:4 (v/v) ratio of
sample to sugar and incubated at room temperature for at least 30 minutes in the dark.
Granulocytes were prepared as seen in section 10.8.2. Granulocytes were fixed with
paraformaldehyde, washed and diluted to a final concentration of 10 x 106/ml. The
remainder of the set up was the same as the GIFT bioassay (refer to section 10.8.4).
The sample used in this set up corresponded to the previously prepared sample-sugar
mixture.
room temperature for 30 minutes in the dark. The plate was washed again to remove
any excess streptavidin-FITC and 100 µl of Bornes EDTA/BSA was added to each of
the wells. The contents of the wells were mixed well and transferred into labelled flow
cytometer tubes containing 250 µl of Bornes EDTA/BSA (control tubes contained 500
µl). These tubes were vortexed prior to being read on the flow cytometer.
9.8.5 Sugar blocking GIFT
___________________________________________________________________ 205
Chapter 9 Experimental _____________________________________________________________________________________
9.9 Mass spectroscopy
single quadruple electrospray mass spectrometer (Fison instruments) was used to
etermine the molecular weight of the eluted proteins from the G.robusta. As some of
the proteins were isolated using buffers, each sample was prepared by running it
rough a small plug of C-18 resin (1ml bed) in water and eluting with 50% acetonitrile
in water. This removed any salt found in the sample that could affect the mass
pectrometer. The samples were freeze-dried and resuspended in 50%
cetonitrile/water (total volume was 200 µl). The spectrometer was set to positive
lectrospray and 3 µl of sample was directly injected into the spectrometer using 50%
acetonitrile/water. The Masslynx icromass) software program was
used to analyse the results.
9.10 NMR Spectroscopy
9.10.1 NMR measurements
All 1D and 2D spectra were recorded using a Varian INOVA 600MHz spectrometer at
either 303 K or 288 K. The GR1 protein was resuspended in 18% CD3CN/H2O or in
99.99% D2O at pH 3.5. All spectra were analysed using the program VNMR. Water
suppression was achieved using a WATERGATE sequence in the TOCSY and NOESY
experiments while a presaturation sequence was used for the suppression in the DQF-
COSY experiment. The mixing times for the NOESY spectra were 125ms and 200ms
for the D2O sample and 75 ms, 125 ms and 200 ms for the H2O sample. NOESY
spectra were recorded at 288 K and 303 K. TOCSY spectra had a mixing time of 80 ms
A
d
th
s
a
e
NT (version 3.4; M
___________________________________________________________________ 206
Chapter 9 Experimental _____________________________________________________________________________________
and were run at 288 K and 303 K for both D O and H O samples. All spectra obtained
were processed by a sine-bell window using a 60° phase shift in the F2 and F1.
lowly exchanging amides protons were determined by dissolving the protein in
leted over a period of 16 hours
mediately after the solvent was added to the protein and run again 48 hours after to
exchanged.
tereospecific assignments of the β protons were determined by identifying the HN-Hβ
and Hα-Hβ connectivities. Each of the stereospecific assignments was added to the
CALIBA macro. The disulfide bonds between Cys11-Cys23, Cys13-Cys21 and Cys32-
Cys46 were also added to the macro. Hydrogen bonds found between Thr14-Arg20 and
Val12-Arg22 from the slowly exchanging amide experiment was also added to the
macro by restraining the HN-O connectivity to 2.4 Å. The modified CALIBA macro
was run in DYANA (version 1.4) to generate the distance restraint file to be used in the
2 2
S
99.99% D2O at pH 3.5 and the experiments were run at 303 and 288 K. One-
dimensional and TOCSY experiments were comp
im
ensure all of the amides had
9.10.2 NMR distance restraints
The peaks from the TOCSY, DQF-COSY and NOESY experiments were tabulated
using the software program XEASY. Rectangular volume integration was used on the
NOESY spectra (with a mixing time of 200 ms) to select peaks and provide volumes for
each peak. The CALIBA macro within DYANA was modified and used to convert the
peak volumes into distance restraints. A series of commands were used to include the
disulfide bonds, the stereospecific assignments and the hydrogen bonds in the CALIBA
macro.
S
___________________________________________________________________ 207
Chapter 9 Experimental _____________________________________________________________________________________
generation of the structures. The CALIBA macro used for each of the experiments is
outlined in Appendix D.
set number of steps. For all structures
generated throughout this work, the following procedure was used and a full list of the
nd 1000 steps of minimisation. The calculation began with
00 molecular dynamic steps at a temperature of 8.0 (temperature was a measure of the
value. This overview file also included the restraints that were violating (upper and
lower limits and vdw) over the set limits, which was useful in determining where
potential problems could arise.
Twenty structures with the lowest target function value were visualised using the
program MOLMOL and the RMSD values were calculated within this program using
the converged structures. The stereochemical quality of each of the structures was
determined using the program PROCHECK (Laskowski et. al., 1993).
9.10.3 Structure calculations
DYANA version 1.5 was also used to generate the structures of the protein (Güntert et.
al., 1997). The macro ANNEAL was modified to include the distance restraints
generated from CALIBA and to perform a
protocols used for ANNEAL are outlined in Appendix D.
One hundred structures were generated per DYANA calculation using a total of 10000
molecular dynamics steps a
8
target function units per degree of freedom) which was followed by a slow cooling stage
for 9200 steps of molecular dynamics until it reached a temperature of 0.0. Finally the
structures were subjected to 1000 steps of minimisation. The resulting structures were
tabulated in the overview file that listed the structures with the lowest target function
___________________________________________________________________ 208
Chapter 9 Experimental _____________________________________________________________________________________
9.10.4 Further refinement
Twen of th e ith e lo st ta et function values
were further refined using the program S YL Th isu br es e a d to
each ctur d a d DY A w s als added. A total of
8000 steps of conjugated gradient min isation using the Tripos force field were
appli o each f th s r T firs wo s s co aine 000
steps of minim tion and t inal step tain 40 tep Th tal energi er
compared and the stereochemical qua determined for each structure using
PRO ECK w ere t re h r d 2.0 Å
of the generated structures
ty e structures generat d from DYANA w th we rg
YB ®. e d lfide idg wer dde
stru e and istance restr ints generate in AN a o
im
ed t o e 20 tructu es over three steps. he t t tep nt d 2
isa he f con ed 00 s s. e to es w e
lity was
CH h he solution for t ese st uctures were define as .
___________________________________________________________________ 209
Appendices ____________________________________________________________________________________
Appendices
___________________________________________________________________ 210
Appendix A ____________________________________________________________________________________
Appendix A Methodology used to determine the full amino acid
sequence of the GR1 protein from Grevillea robusta.
A-1 RNA extraction
The developing seeds of G.robusta were removed from the fruit and seed coat and
ground using a mortar and pestle under liquid nitrogen and the total RNA was extracted
using the method outlined by Cheng et. al., 1993. Absorbance readings were taken at
the wavelengths of 260 and 280nm to determine the purity and quantity of RNA
present. Electrophoresis on an RNase free 2% NuSieve agarose gel allowed the RNA to
be visualised. The RNA sample was treated with RQ1 RNase-free DNase according to
the manufacturer’s instructions to ensure the sample did not contain any DNA
contamination. The RNA was recovered using ethanol precipitation and the samples
This information has been taken from the final assessment for a coworker at the ARCBS
as part of her Masters degree (Clague et. al., 1999).
immediately placed into liquid nitrogen where they were stored. The seeds were finely
were resuspended in 50µl of RNase-free water and stored at -80°C.
___________________________________________________________________ 211
Appendix A ____________________________________________________________________________________
A-2 3’RACE
The N-terminal sequences obtained from t G.robusta seeds
were used to generate 12 degenerative, inosine containing oligonucleotide primers for
the 5’en he 3 E oligo
(dT) prim us rformed
using Superscript™ (II) RNase H- Reverse Transcriptase and the oligo (dT)20 primer
with 5µg of purified RNA in 20µl reaction.
The 3’R E R s fo d using 100 of c m tu h ontained
50mM KCl, 10mM Tris pH 8.3, 1.5mM MgCl , 0.2mM of each dNTP, 20pmol primer
B, 10pm o pliTaq
Gold™
The rea minutes and paused to add the
AmpliTa u and this
involved n 5°C for
30 secon r econds.
The next 10 cycles involved: 94°C for 30 seconds, 40°C for 30 seconds, 72°C for 30
ose gel and ethidium bromide. The DNA
d from the gel using the QIA quick gel extraction kit.
the proteins isolated from he
d of the cDNA. These primers are shown in Figure A-1. T ’RAC
er was ed for the unknown 3’end. The first strand of cDNA was pe
AC PC wa per rme µl rea tion ix re w ich c
2
ol 3’RACE primer, 1µl cDNA (equivalent to 1µg) and 2.5U f Am
DNA polymerase.
ction mixture was heated to 94°C for 10
q Gold™ DNA polymerase. The touchdown PCR protocol was sed
the first 30 cycles to consist of the following: 94°C for 30 seco ds, 5
ds with the temperature decreasing 0.5°C per cycle and 72°C fo 30 s
seconds and finally 72°C for 7 minutes. The DNA products were visualised
electrophoretically using a 2% NuSieve agar
products were purifie
___________________________________________________________________ 212
Appendix A ____________________________________________________________________________________
G2 G G A GGI GGI GAG GAG GCI GA3’ B --- --- --G --A --- --- C D
E D/E D V V T T S C I P P E GAI GAI GAT GTT GT3’
G CCI AAC ATI TGI AT
--- GAT TGI TGG
L --- --- --- --c --- ---
3'RACE universal primer 5'CGCCTAGG(T)17 3'
igure A-1: A list of the degenerative primers designed from the N-terminal
equencing results. The N-terminal sequences are in bold.
E E A D W C
--- --- --A --G --- --- --- --- --A --A --- ---
G2..cont
F --- --- --- --C ---
G3 C L P N I C S S D L D 3’
H --- --T --- --- --- G4 S I P E A D C W R C T D I TCT ATI CCI GAA GCI GA3’ J --C --- --- --- --- K CCI GAA GCI
F
s
___________________________________________________________________ 213
Appendix A ____________________________________________________________________________________
the sequencing results obtained from the 3’RACE experiment
were designed and these primers are shown in figure A-2.
GR1 5' G C C A A C A G G T T C A G A A G A T 3' GR2 5' G G C T A G G A C C A C A C C A T G 3'GR3 5' C C G G A G C A G T A G A G G A G A C 3'
igure A-2: The specific pri ers deve ped from the 3’ resu
imer l GR1 w used he o fir of cDNA
ma urer’s ctions). Two 5’RACE PCR reactions were completed
2 R3 pri indiv ly. tio ure s what was
utlined in the manufacturer’ instructions. The CR pr us 4°C for 10
inutes which was followed by 35 cycles of: 94°C for 30 seconds, 55 r 30 seconds,
2°C for 60 seconds and finally 72°C for 7 minutes. The DNA products produced
alise e 2% eve a e g se cts ified using
uick ractio
A-3 5’RACE
The 5’RACE system for rapid amplification of cDNA ends was used (Gibco). Three
primers selected from
G AG G T T
T A T
F m lo RACE lts.
The pr abeled as for t synthesis f the st strand
(following nufact instru
using the GR and G mers idual The reac n mixt used wa
o s P otocol ed was 9
m °C fo
7
were visu d on th NuSi garos el and the produ were pur
the QIA q gel ext n kit.
___________________________________________________________________ 214
Appendix A ____________________________________________________________________________________
A-4 Sequencing of the cDNA
he purified DNA products were re run on the 2% NuSieve agarose gel with
be
ined. The final PCR reaction volume of 20 µl included 200ng of template DNA,
rd primer and 8 µl of Big Dye Terminator reaction mix. The control
action contained 200ng of pGEM-3Zf(+) as template DNA ad 3.2 pmol of M13 as the
e to 25 cycles at 96°C for 10 seconds, 50°C
oducts were transferred to 1.5 ml eppendorf tubes that contained 2
hanol/5% isopropanol (v/v) and
mples were centrifuged at 21000 x g at 4°C for
The precipitated DNA pellet was washed
re centrifuged again in a microfuge at
aximum speed for 15 minutes. The supernatant was removed and the tubes were
minute to dry the sample. These samples were sequenced for a
located at the Queensland Institute of Medical Research.
e resulting sequence.
T
quantitative molecular weight markers, which allowed to amount of DNA present to
determ
3.2 pmol of forwa
re
forward primer. Both sampl s were subjected
for 5 seconds and 60°C for 4 minutes.
The resulting PCR pr
µl of 3 M sodium acetate pH 4.6 and 50 µl of 95% et
incubated on ice for 15 minutes. The sa
30 minutes and the supernatant was removed.
with 500 µl of 70% ethanol and the samples we
m
incubated at 90°C for 1
fee on the ABI Prism system
Figure A-3 shows th
___________________________________________________________________ 215
Appendix A ____________________________________________________________________________________
35 AGA GTG CAA GTG ATC AGA GAT CGA TCG AGA GAG 71
A V A K V A L M I T L 12 CT GTT GCT AAG GTG GCG TTG ATG ATA ACA CTA 107
L L F V A T L P A P 24
CTG CCT GCT CCA 143
S T A T S N P F G P F R 36 179
P S G G E E A D W C E D 48 CCA AGT GGT GGA GAG GAA GCT GAC TGG TGC GAA GAC 215
TGT GTT TGC ACA AGA TCA ATT CCT CCT CGC TGT GTT 251
C T D S S V C T K C V C 72 TGC ACT GAT TCT TCG GTG TGA ACC AAA TGT GTT TGC 287
Y L T V P A A M R P Y C 84 TAC CTA ACT GTA CCT GCT GCA ATG AGG CCT TAT TGT 323
E S M A S R F D A F C P 96 GAG TCT ATG GCT TCC AGA TTC GAT GCC TTC TGC CCC 359
I G S L Q S Y N 104 ATT GCC TCT CTT CAA TCC TAC AAC 383
TGA TCG ATG AGC TCA ACA GAA CCC TAA ATA GTC TCC 419TCT ACT GCT CCG GCT GTC AAC CAT GGT CGT GGT CCT 455AGC CAG CTT ATA TAT GCA GTT CTT TTC TAC TTT ATG 491TCT TGT ATT TCT TCT CTT AGT TTC ATC TTC TGT AAC 527CCA GTT GGC AGT TGT TAG CGA AAG TGG CTA ACA ACT 563AGT TTG TTG ATA GTT GAT AAT AAA GAG GAG ATT TTC 599ATA AAA AAA AA 610
Figure A-3: The complete amino acid sequence of the GR1 protein from the seeds
f G.robusta. The start of the N-terminal sequence is highlighted in red. The one letter code is used for the
GG GGG GGG ACC GTG TGT TTG TGT AAA CTG TAG GTG TGA
M ATG G
M V ATG GTG TTG CTC TTC GTA GCA ACA
TCG ACT GCA ACA AGC AAC CCG TTC GGG CCG TTC AGA
C V C T R S I P P R C R 60
o
amino acid residues.
___________________________________________________________________ 216
Appendix B
______________________________________________________________________________
R1 protein isolated from the seeds of G.robusta.
B-1 Trypsin and Chymotrypsin inhibition assays
he protease inhibition assays were carried out using the instructions provided by the
anufacturer (Roche). This assay used casein, resorufin-labeled, universal protease
ubstrate. The inhibition constants were calculated at pH 7.8 and at 24°C. Data was
nalysed using the GraphPad Prism software. The inhibition curves are shown in Figure
-1 for (A) trypsin and (B) chymotrypsin.
igure B-1: The inhibition curves for the GR1 p
Appendix B Enzymatic inhibitory studies of the G
T
m
s
a
B
A
F
________________________________________
B
rotein.
___________________________ 217
Appendix C
______________________________________________________________________________
Appendix C Experimental Random coil values
The experimental random coil values used are shown below and these values were taken
from Wüthrich, 1986.
Residue NH αH βH Others
Glycine Gly, G 8.39 3.97 Alanine Ala, A 8.25 4.35 1.39 Valine Val, V 8.44 4.18 2.13 Isoleucine Ile, I 8.19 4.23 1.9 γCH3 0.97, 0.94 γCH2 1.48, 1.19 γCH3 0.95 δCH3 0.89 Leucine Leu, L 8.42 4.38 1.65, 1.65 γH 1.64 δCH3 0.94, 0.90 Proline Pro, P 4.44 2.28, 2.02 γCH2 2.03, 2.03 δCH2 3.68, 3.65 Serine Ser, S 8.38 4.5 3.88, 3.88 Threonine Thr, T 8.24 4.35 4.22 γCH3 1.23 Aspartic acid Asp, D 8.41 4.76 1.84, 1.75 Glutamic acid Glu, E 8.37 4.29 2.09, 1.97 γCH2 2.31, 2.28
εCH2 3.02, 3.02 εNH3- 7.52 Arginine Arg, R 8.27 4.38 1.89, 1.79 γCH2 1.70, 1.70 δCH2 3.32, 3.32 NH 7.17, 6.62 Asparagine Asn, N 8.75 4.75 2.83, 2.75 γNH2 7.59, 6.91 Glutamine Gln, Q 8.41 4.37 2.13, 2.01 γCH2 2.38, 2.38 δNH2 6.87, 7.59 Methonine Met, M 8.42 4.52 2.15, 2.01 γCH2 2.64, 2.64 εCH3 2.13 Cysteine Cys, C 8.31 4.69 3.28, 2.96 Tryptophan Trp, W 8.09 4.7 3.32, 3.19 2H 7.24 4H 7.65 5H 7.17 6H 7.24 7H 7.5 NH 10.22 Phenylalanine Phe, F 8.23 4.66 3.22, 2.99 2,6H 7.3 3,5H 7.39 Tyrosine Tyr, Y 8.18 4.6 3.13, 2.92 2,6H 7.15 3,5H 6.86 Histidine His, H 8.41 4.63 3.26, 3.20 2H 8.12 4H 7.14
Lysine Lys, K 8.41 4.36 1.85, 1.76 γCH2 1.45, 1.45 δCH2 1.70, 1.70
___________________________________________________________________ 218
Appendix D
______________________________________________________________________________
___________________________________________________________________ 219
ppendix D Structure Calculations
-1 CALIBA and ANNEAL macros used to generate the DYANA structures
ALIBA_B.dya
ar # deletes all distance constraints ad seq SO2protein_mod.seq # read sequence ad prot /userdata/people/sarahk/xeasy_files/revnoesy.prot # read proton list
y_files/REVNOESY.peaks assigned integrated # read eak list
istance unique # keep strongest constraint for each write upl caliba_A.upl ssbond 8-23 11-57 13-21 29-34 32-46 atoms stereo HB2 7 8 11 16 21 34 35 36 42 48 49 53 54 56 toms stereo QG1 38 istance make 2.4 14 HN THR, 20 O ARG
weight=1.0 distance make 2.4 12 HN VAL, 22 O ARG weight=1.0 distance modify write upl caliba_B.upl # save upper limits write lol caliba_B.lol # save lower limits
ANNEAL_GR1D.dya
read seq SO2protein_mod.seq read upl caliba_B.upl read lol caliba_B.lol distance modify write lol GR1protein_D.lol write upl GR1protein_D.upl random_all 100 forall parallel anneal thigh=8.0 tend=0.0 steps=10000 highsteps=800 minsteps=1000 structure sort cutNo = 0 do i 1 100 if (tf (i) <= 2000) then cutNo = i end if end do structure select 1..cutNo write pdb GR1_D.pdb all
A
D
C
distance clerereread peaks /userdata/people/sarahk/xeaspcaliba d
ad
Appendix D
______________________________________________________________________________
pdb structure select ut_tf := 50 ut_upl := 0.5
overview GR1_D structures=100 ang cor hbond vdw
D-2 Stereochemical Quality of the DYANA Structures
PROCHECK was used to determine the quality of the structures generated from
DYANA. A summary of these results is shown below.
DYANA generated structures
GR_3 2.14 32.7 48.1 17.3 1.9 80.8
GR_2 1.82 17.3 59.6 19.2 3.8 76.9GR_15 3.06 28.8 46.2 19.2 5.8 75GR_16 3.08 21.2 46.2 28.8 3.8 67.4
GR_13 2.93 15.4 57.7 23.1 3.8 73.1
GR_10 2.89 21.2 40.4 30.8 7.7 61.6GR_12 2.93 25 50 21.2 3.8 75GR_18 3.37 36.5 32.7 21.2 9.6 69.2GR_5 2.52 28.8 44.2 25 1.9 73GR_6 2.79 26.9 51.9 17.3 3.8 78.8GR_19 3.43 26.9 46.2 21.2 5.8 73.1
write_all GR1_D
cccut_lol := 0.5 cut_vdw := 0.2 hb_len := 2.4 hb_ang := 0.610865
DYANA Procheck results before minimisationTF core allow gener diallow c + a
GR_14 3.01 26.9 44.2 25 3.8 71.1
GR_4 2.37 30.8 46.2 19.2 3.8 77GR1_1 1.78 34.6 44.2 15.4 5.8 78.8
Figure D-1: PROCHECK results of the
GR_20 3.44 28.8 40.4 25 5.8 69.2
Av 2.82 26.63 46.64 21.64 5.08 73.3
GR_8 2.88 23.1 51.9 23.1 1.9 75GR_7 2.86 30.8 48.1 15.4 5.8 78.9GR_11 2.90 26.9 44.2 19.2 9.6 71.1GR_17 3.36 28.8 48.1 19.2 3.8 76.9
GR_9 2.89 21.2 42.3 26.9 9.6 63.5
___________________________________________________________________ 220
Appendix D
______________________________________________________________________________
___________________________________________________________________ 221
s after (A)
000 steps and (B) 4000 steps.
D-3 Stereochemical Quality of the SYBYL® minimised structures
Procheck results after 2000 steps of minimisation
GR_14 64.35 42.3 40.4 15.4Energy Core Allow gener disall c + a
1.9 82.7GR_3 65.00 51.9 42.3 3.8 1.9 94.2GR_4 75.63 44.5 36.5 13.5 5.8 81GR1_1 79.71 42.3 40.4 7.7 9.6 82.7GR_2 80.43 38.5 46.2 7.7 7.7 84.7
GR_16 83.58 32.7 50 7.7 9.6 82.7.7 48.1 7.7 11.5 80.8.3 44.2 5.8 7.7 86.5
GR_11 91.68 34.6 50 7.7 7.7 84.6GR_17 91.73 40.4 46.2 9.6 3.8 86.6GR_13 94.73 30.8 44.2 9.6 15.4 75
GR_12 97.95 32.7 48.1 13.5 5.8 80.8
GR_5 110.39 44.5 38.5 13.5 3.8 83GR_6 111.80 44.2 40.4 7.7 7.7 84.6GR_19 119.59 48.1 26.9 15.4 9.6 75GR_20 129.70 32.7 48.1 9.6 9.6 80.8
Av 92.96 38.30 44.05 9.82 7.88 82.34
GR_15 83.58 32.7 50 7.7 9.6 82.7
GR_8 86.41 32GR_7 87.23 42
GR_9 97.49 28.8 50 13.5 7.7 78.8GR_10 97.83 34.6 46.2 7.7 11.5 80.8
GR_18 110.30 34.6 44.2 11.5 9.6 78.8
SD 17.13 6.45 5.76 3.31 3.36 4.21
Procheck results after 4000 s teps of minimisation
Energy Core allow gener disall c + aGR_3 53.332 51.9 42.3 3.8 1.9 94.2GR_14 53.8467 46.2 36.5 15.4 1.9 82.7GR_2 54.84097 38.5 48.1 3.8 9.6 86.6
GR_8 71.2816 36.5 46.2 5.8 11.5 82.7GR_15 71.8524 34.6 46.2 9.6 9.6 80.8GR_16 71.8524 34.6 46.2 9.6 9.6 80.8GR_11 72.3013 30.8 55.8 5.8 7.7 86.6GR_7 74.2422 40.4 48.1 3.8 7.7 88.5
GR_10 77.5409 30.8 50 7.7 11.5 80.8GR_9 79.2774 30.8 51.9 9.6 7.7 82.7GR_12 88.6027 32.7 44.2 17.3 5.8 76.9
GR_20 95.2538 36.5 42.3 11.5 9.6 78.8
45.005 8.645 8.165 83.18SD 15.074 6.218 5.360 4.072 3.350 4.33
GR_4 61.0314 42.3 42.3 9.6 5.8 84.6GR_1 64.5039 38.5 48.1 3.8 9.6 86.6
GR_13 74.9967 28.8 50 5.8 15.4 78.8GR_17 76.3022 42.3 44.2 7.7 5.8 86.5
GR_6 90.5669 46.2 40.4 5.8 7.7 86.6GR_5 92.2646 44.2 36.5 15.4 3.8 80.7
GR_18 97.2014 34.6 46.2 9.6 9.6 80.8GR_19 107.1231 42.3 34.6 11.5 11.5 76.9
AV 76.411 38.175
Figure D-2: PROCHECK results of the SYBYL® minimised structure
2
Appendix D
______________________________________________________________________________
PROCHECK results after 8000 steps of minimisationEnergy core allow gener disall c + a
GR_14 40.6034 44.2 38.5 13.5 3.8 82.7GR_2 41.6047 36.5 48.1 5.8 9.6 84.6
___________________________________________________________________ 222
Figure D-3: PROCHECK results after 8000 steps of minimisation using SYBYL®.
GR_4 46.0056 38.5 44.2 11.5 5.8 82.7
7.7 80.77.7 84.6
GR_10 63.0637 25 53.8 9.6 11.5 78.8GR_7 63.2059 48.1 40.4 3.8 7.7 88.5GR_15 66.228 34.6 48.1 7.7 9.6 82.7
GR_5 80.4959 44.2 38.5 11.5 5.8 82.7
GR_19 94.0553 38.5 38.5 11.5 11.5 77
63.27 37.40 46.06 8.06 8.46 83.4615.60 6.99 5.51 3.78 3.08 4.47
GR_3 44.78 51.9 42.3 3.8 1.9 94.2GR_11 45.1107 38.5 50 3.8 7.7 88.5
GR_1 49.1346 34.6 53.8 1.9 9.6 88.4GR_8 58.0681 30.8 51.9 5.8 11.5 82.7GR_9 60.6977 26.9 53.8 11.5GR_17 61.6472 42.3 42.3 7.7
GR_16 66.228 34.6 48.1 7.7 9.6 82.7GR_13 67.0577 28.8 51.9 3.8 15.4 80.7GR_20 72.8701 34.6 40.4 13.5 11.5 75GR_6 75.9208 44.2 44.2 3.8 7.7 88.4
GR_12 81.4724 32.7 50 11.5 5.8 82.7GR_18 87.2117 38.5 42.3 11.5 7.7 80.8
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