Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

EXPRESSION AND CHARACTERISATION

OF THE N-TERMINAL HALF

OF HUMAN LACTOFERRIN

his thesis is submitred to Massey University as partial fulfilment of the requirements

for the degree of Doctor of Philosophy in Biochemistry.

Catherine Louise Day

January, 1993

ACKNOWLEDGEMENTS

Throughout the duration of this thesis many people have offered help and listened to my

problems. It is not possible to list everybody and I hope that if your name has been omitted you

will accept my thanks here.

Firstly I would like to thank my two supervisors, Dr. John W. Tweedie and Prof. Ted N.

Baker, for their encouragement, helpful discussions, patience and friendship. They have both

shared the successes and failures of this work and for that I am grateful. I would especially

like to thank them both for helping with the preparation of this thesis.

Others have also contributed significantly to the work presented in this thesis. In particular I

would like to thank Kathryn S to well who helped me with much of the molecular biology and

provided the initial clones which made this work possible. Thank you for your help and

friendship Kathryn. My thanks are also extended to Gill Norris who set up many of the

preliminary crystallisation trials and helped with the characterisation of the crystals. Thank you

also for always providing a sympathetic ear Gill. Bryan Anderson is thanked for his help with

the computing, without your help I would have smashed the computer long ago!. I also wish to

thank Clyde Smith for helping with processing the CAD4 data, preparation of the ESR figures

and for many helpful discussions. My thanks are also extended to Heather Baker for her helpful

suggestions in characterising the proteins, encouragement and cheerfulness.

I also appreciate the work of Ted, Bryan, Gill and Heather who collected data for me at the

Photon Factory, Tskuba, Japan. The Molecular Structure Corparation (USA) is also

acknowledged for collecting a data set

My thanks are also extended to Dr. Ross T.A. MacGillivray (University of British Columbia)

/ who allowed me to spend two months in his laboratory where I learnt the art of tissue culture.

I also wish to thank all past and present members of the Twilight Zone in particular Paul

Mead, Bhav Sheth, Heather Bain, Joseph Bateson, Hale Nicholson, Cherie Stayner and

Samantha Shaw who have shared their time with me and helped make life more enjoyable.

I am indebted to the University Grants Committee of New Zealand for awarding me a scholarship

which made this work possible.

Lastly I would like to thank my family and friends for their support and encouragement

Thank you everybody.

CONTENTS

ABSTRACT ABBREVIATIONS LIST OF TABLES LIST OF FIGURES

1 . 1 1.2 1.2. 1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.2

1.4 1.5 1 . 6

Chapter One

INTRODUCTION

Distribution and regulation of lactoferrin gene expression Biological roles of lactoferrin Bacteriostatic and bactericidal properties of lactoferrin Lactoferrin as an iron carrier Lactoferrin as a growth promoter Lactoferrin and inflammation Lactoferrin: oxidant or antioxidant ? Lactoferrin and myelopoiesis Structurt: of lactoferrin Primary structure Tertiary structure Binding properties of lactoferrin Fragmen� studies of lactoferrin Perspectives and aims of this project Perspectives Aims

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Part A CLONING, CHARACTERISATION AND MUTAGENESIS OF THE N-TERMINAL HALF OF HUMAN LACTOFERRIN

A l. 1 A1.2 A l.2.1 A1 .2.2

Part A : Chapter One

MATERIALS AND METHODS

Material�: and Reagents General methods for the manipulation of DNA and RNA General precauti�ns for handling DNA and RNA Quantitation of DNA and RNA .

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25

25

A l.2.3 A1.2.4 A1.2.S A1.2.6 A1.2. 7 A1.2.8 A1.2.9 A1.2.10

A l.2.11 A1.2.12 A1.2.13 A1.2.14 Al.2.1S Al.2.16 A l.2.17 A1.2.18 A l.2.19 A l.2.20

Al.2.21 A1.2.23 A1.3 A1.3.1 A l .3.2 A1.3.3 A l.3.4 A l.4 A l .4.1

A1.4.2 A1.4.3 A l .4.4

Phenol I chloroform extraction of DNA Ethanol precipitation of DNA Maintenance and storage of bacterial strains Preparation. and transformation of competent cells Digestion of DNA with restriction endonucleases Agarose gel electrophoresis of DNA Autoradiography Preparation of plasmid DNA Small scale preparations Large scale preparations Isolation of phagemid ssDNA DNA Ligations Isolation of DNA fragments from agarose gels End filling of cohesive ends Phosphorylation of blunt ended DNA fragments Dephosphorylation of linearised plasmid DNA DNA sequencing Agarose gel electrophoresis of RNA Transfer of RNA to nitrocellulose (RNA blotting) Labelling of cDNA probes with 32p Hybridisation using cDNA probes Prehybridisation Hybridisation Washing Preparation and purification of oligonucleotides Amplification of DNA sequences by PCR Mutagenesis of cloned cDNA Preparation of uracil-containing phagemids Extraction of phagemid DNA Phosphorylation of the oligonucleotides Annealing and extension of the primer Expression of cloned LfN cDNA Maintenance of cells in tissue culture media Preparation of tissue culture reagents Freezing und thawing of cells PaSsage of cells Transfection and selection of BHK cells Large scale growth of cells in roller bottles Preparation of DNA from BHK Cells

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A1.4.S Preparation of total cellular RNA from BHK cells 36

A l .S Analysis of recombinant proteins 36

A l .S .1 SDS-polyacrylamide gel electrophoresis of proteins 36

A l .S .2 Staining and destaining polyacrylamide gels 36

A l .S .3 Preparation of antibodies against human lactoferrin 37

A l .S .4 Immunoprecipitation of human lactoferrin 37

A l .S .S 35S-Labelling of newly synthesised proteins 37

A l .S .6 Protein blotting 38

A l .S . 7 Purification of recombinant lactoferrins 39

A l .S .8 Determination of protein concentration 39

A l .S .9 Concentration of protein samples 39

A l .S .10 Deglycosylation of lactoferrin 40

A l .S .11 Spectroscopy 40

A l .S .1 2 Iron release from lactoferrin 40

Part A : Chapter Two

CLONING AND EXPRESSION OF THE N-TERMINAL HALF

OF HUMAN LACTOFERRIN

A2.1 A2.2 A2.2.1

A2.2.2

A2.2.3

INTRODUCTION RESULTS PCR synthesis and cloning of the 1.1 kb cDNA encoding LfN Preparation of templates Synthesis of the cDNA by PCR Restriction enzyme analysis of the 1.1 kb PCR fragment Cloning of the 1.1 kb PCR product Preliminary sequence analysis of the 1.1 kb insert Cloning of LfN cDNA into the expression vector pNUT Preparation of pNUT for use as a vector Cloning of the LfN cDNA into pNUT Expression of LfN in transfected BHK cells Transfection and selection of BHK cells Analysis of BHK cell genomic DNA Analysis of BHK cell RNA Further analysis of the pNUT clones

0 , <• I • -• ; \..·.'' � I ·:� .,,'

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44 45

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A2.2.4

A 2.3

Construction of a new cDNA Synthesis and cloning of cDNA nwnber 2! Reassessment of the results obtained so far Subcloning to remove the e"or Cloning into pNUT Transfection of BHK cells and analysis of the RNA Analysis of the cell media DISCUSSION

Part A: Chapter Three

CHARACTERISATION OF LfN

A3.1 INTRODUCTION A3.2 RESULTS A3.2.1 Purification of recombinant LfN A3.2.2 Characterisation of recombinant L fN A3.2.2.1 Absorption spectrum A3.2.2.2 N-termin�l sequence analysis A3 .2.2.3 Glycosylation A3.2.2.4 Spectroscopy A3.2.2.5 Metal binding and release A3.3 DISCUSSION

Part A : Chapter Four

M UTAGENESIS STUDIES ON HUMAN LACTOFERRIN

A4.1

A4.2 A4.2.1

INTRODUCTION Aspartic acid 60 Arginine 121 Glycosylation of lactofe"in Mutagenesis protocols RESULTS Procluction of a mutant Cloning into p'IZ and_preparation of the template phage Titration and extraction of the phage Synthesis of the complementary strand Analysis of the reaction products

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A4.2.2 A4.2.3

A4.2.4

A4.3

Part B

Bl.l

B1.2

B1.3 B1.3.1

B 1.3.2

B1.4

Bl.S

Selection and complete sequence analysis of the mutants Cloning and expression of the mutants Glycosylation site mutants Asparagine 137 Asparagine 137 I asparagine 478 double mutant Binding site mutants Aspartic acid 60 Arginine 121 Arginine 121 I Aspartic acid 60 DISCUSSION

CRYST ALLOGRAPHIC ANALYSIS OF LfN

Part B : Chapter One

METHODOLOGY AND PROGRAM DESCRIPTION

Crystal growth Crystal growth Data collection CAD4 data collection Image plate data collection Rigaku - R axis axis data collection Data processing CAD4 data processing PREPRO FIT'C3 REDUCER Image plate data processing Convmt Image Weis Weisdatal Scaling and merging of data INTAV3 INTAV4 "Structure solution

- Multiple isomorphous replacement (MlR)

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99 99 99 99

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B1.6

B1.7

3.4.1 B2.2 B2.2.1 B2.2.2

B2.2.3

3.4.5

B3.1 B3.2 B3.2.1

B3.2.2

B3.2.3

Molecular replacement (MR) ALMN Tsearch Protein structure refinement TNT - the restrained least squo.res refinement package. Map calculations and model building

2F obs - F calc I Fobs - F CaJc FRODO - the model building package

Part B : Chapter Two

CRYSTALLISATION OF LfN

INTRODUCTION RESULTS Crystallis.,ation of native LfN Crystallisation of deglycosylated LfN FeLfN ApoLfN Characterisation of the FeLfN and DG FeLfN crystals Photography of FeLfN crystals Photography of DG FeLfN crystals Photography of ApolfN crystals DISCUSSION

Part B : Chapter Three

FeLfN STRUCTURE SOLUTION AND ANALYSIS

INTRODUCTION RESULTS CAD4 data collection and processing Data collection Data processing Structure solution in Pl The models The rotation functio� The translationfunction Refinement of the structure in Pl

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Rigid body refinement 119

B3.2.5 Determination of the correct space group 120

B3.2.5 Collection and processing of the high resolution data 122

Collection of the data 122

Processing the data 123

Merging the image plate data 125

Analysis of the C2 data set 126

B3.2.6 Structure, solution in space group C2 126

B3.2. 7 Refinement of the FeLfN structure in space group C2 127

Criteria for the inclusion of additional sidechains 128

Positioning residues 313-333 129

Solvent structure 130

B3.2.8 Quality of the refined model 131

Accuracy of the structure 131

Thermal parameters 134

Dihedral angles 135

B3.2.9 Analysis of the refined structure 138 -

B3.2.9.1 General topology 138

B3.2.9.2 Analysis of the metal· and anion binding sites in FeLfN. 142

B 3 .2.9 .3 Conformational changes in FeLfN 146

Residues 312 -333 146

1 ntermolecular interactions 151

B3.2.9.4 Solvent structure 153

B3.3 DISCUSSION 157

Part B : Chapter Four

ApoLfN DATA COLLECTION AND STRUCTURE SOLUTION

B4.1 B4.2 B4.2.1

B4.2.2

INTRODUCTION RESULTS Data collection and processing Data collection Data processing Solution of the ApoLfN structure The models The rotation ftuzction The translation function Rigid body refinement of the model in space group P4J2J2

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165

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167

B4.2.3 B4.2.4 B4.2.S

B4.3

j Partial refinement of the ApoLfN structure Accuracy of the ApoLfN structure Analysis of the ApoLfN structure Conformation and crystal packing of ApoLfN The binding site in ApoLfN DISCUSSION

Chapter Two

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177

CONCLUSIONS AND FUTURE DIRECTIONS

2.1 2.2 2. 2. 1

2. 2. 2

2. 2. 3

Conclusions and final discussion Future directions Suggestions for future constructs Construction of an expression system for the C-terminal half of human lactoferrin Other iron binding fragments Chimaeric fragments Suggestions for other site specific mutants Glycosylation mutants Meanotransferrin model Complete removal of the iron binding capacity from lactoferrin Hinge movement Altered metal and anion binding capacity Further crystallographic and analytical studies

; Crystallographic studies Functional studies

REFERENCES

APPENDIX 1 APPENDIX 2 APPENDIX 3 APPENDIX 4 APPENDIX s APPENDIX 6

, APPENDIX 7

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inside back cover

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1

ABSTRACT

Lactoferrin is an 80 kDa iion binding protein which is found in human milk and other exocrine

solutions. Each molecule contains two metal binding sites which each bind a single iron atom

with high affmity. The properties of the two sites are slightly different In an attempt to more

fully understand the nature of these differences a construct for the high level expression of the N­

terminal half of human lactoferrin (LfN) has been constructed and protein expressed from this

construct has been purified and characterised. Characterisation of the recombinant protein has

shown that the signal peptide is correctly removed from LfN and that anN-linked carbohydrate

. moiety is added to LfN. LfN has been shown to bind one iron atom and the spectral properties

are very similar to those of Fe2Lf. The most significant difference between hLf and LfN is in the

pH stability of iron binding. Iron is released from LfN 2 pH units higher than from hLf.

In an attempt to understand the bases for this difference a structural analysis of LfN was initiated.

Using deglycosylated protein high quality crystals of both iron free and iron saturated LfN have

been grown. The structures of both FeLfN and ApoLtN have both been solved by molecular

replacement using the coordinates from theN-lobe of Fe2Lf as the starting model.

The structure of FeLfN has been refined using data between 8.0 and 2.0 A. The current model

has good geometry and is believed to accurately represent the structure of FeLfN. The structure

of FeLfN provides the highest resolution and most accurate structure of a member of the

transferrin family. Analysis of the structure has shown that the folding pattern and the

environment of the iron atom in FeLfN are very similar to the N-lobe of Fe2Lf although several

differences exist Most of the differences seen are due to the absence of the C-lobe and the

rearrangement of residues 315 - 327. The altered conformation of residues 315 - 327 and the

changes in the solvent accessibility to other residues are believed to be responsible for the

different iron binding and release properties of LfN.

Although the structure of ApoLfN is not complete analysis of this structure has shown that unlike

theN-lobe of intact apo hLf the domains are closed in ApoLfN. The structure of ApoLfN is very

similar to that of FeLfN even though the crystal packing is quite different. In addition although

the protein was believed to be iron free there is some density in the iron site which is unaccounted

for at present. This study continues.

Several mutants of LfN have also been created. These mutants have shown that the carbohydrate

groups attached to lactoferrin probably have a role in folding and secretion of lactoferrin by BHK

cells. Several mutants involving changes to residues involved in metal and anion binding have also been created. These mutants have helped us begin to defme the changes responsible for preventing iron. binding in the C-lobe of melanotransferrin·. In addition the role of arginine 1 21

has been investigated however �er analysis of these mutants is required before the structural changes �sponsible for the different properties can be defmed. . ,-

11

ABBREVIATIONS

A28o Absorbance at 280 run

A-406 Absorbance at 406 run

A-454 Absorbance at 454 run

ApoLf Iron free native lactoferrin

ApoLfN Iron free LfN

ATP adenosine triphosphate

BG background

BHK baby hamster kidney

bLf bovine lactoferrin

bp base pair

BRL Bethesda research laboratories

BSA bovine serum albumin

cDNA complementary DNA

cfu colony forming units

CIF colony inhibitory factor

CML chronic myeloid leukaemia

CM-sephadex carboxymethyl sephadex

cpm counts per minute

C-terminal carboxy terminal

Cu2Lf copper saturated native lactoferrin

CuLfN copper saturated LfN

dATP deoxyadenosine triphosphate

dCfP deoxycytidine triphosphate

DEPC diethylpyrocarbonate

DG deglycosylated

DGLfN deglycosylated LfN

dGTP deoxyguanosine triphosphate

DHFR dihydi-ofolate reductase

DMEM Dulbeco's modification of Eagle's medium

DMF dimethylformamide

DMSO dimethyl sulphoxide

DNA deoxyribonucleic acid

DNase deoxyribonuclease

DTI dithiothreitol

dUTP deoxyuridine triphosphate

EDTA ethylenediamine tetra-acetate

EEO electroendosmosis

ELISA enzyme linked immunoabsorbant assay

ll1

ESR electron spin resonance

F's structure factors

Fca1c calculated structure factors

Fobs observed structure factors

F12 Hams-F12 medium

FCS foetal calf serum

FeLfN iron saturated LfN

Fe2Lf iron saturated native lactoferrin

gDNA genomic DNA

GF/A glass fibre/ A

GM granulocyte macrophage

GM-CSF granulocyte macrophage colony stimulating factor

HBS HEPES buffered saline

HBV hepatitis b virus

HE PES N-2-hyroxethyl piperazine-N-2-ethane sulfonic acid

hGH human growth hormone

hLf human lactoferrin

hTf human transferrin

I intensities

IPA isopropanol

IPTG isopropyl P-D-thiogalactopyranoside

kb kilo base

kDa kilodalton

LB Luria-Bertani

u lactoferrin

LfN the amino terminal half of human lactoferrin

Lfcso 50 kDa carboxy terminal fragment from lactoferrin

LfN30 30 kDa amino terminal fragment from lactoferrin

LMP low melting point

MEM minimal essential medium

MES 2(N-morpholino) ethane sulfonic acid

MIR multiple isomorphous replacement

MPD 2-methyl-2,3-pentanediol

:MR. molecular replacement

MT-1 metallothionein-1

mtx methotrexate

N-terminal amino terminal

NTA nitrilotriacetate

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

PCR

pdb

PEG

pfu

PNGase

RIA RNA

RNase

rRNA

SDS

polymerase chain reaction

protein data bank

polyethylene glycol

plaque forming units

peptide:N-glycosidase

radio immunoassay

ribonucleic acid

ribonuclease

ribosomal ribonucleic acid]

sodium dodecylsulphate

SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis

SSC standard saline citrate

sTf serum transferrin

sTN the amino terminal half of serum transferrin

iv

sTf�BS 35 kDa fragment from the amino tenninal half of human lactoferrin

SV-40 simian virus 40

TA

TAE

TCA

TE Tris

U-DNA

uv X-gal

Tris acetate

Tris acetate EDTA

trichloroacetic acid

lOmMTris, 1 mMEDTA

Tris-(hydroxymethyl) aminomethane

uracil containing DNA

ultraviolet

5-bromo-4-chlor-3-indoyl �-D-galactopyranoside

Table 1

Table 2

Table 3

Table 4

, TableS

Table 6

Table?

Table 8

Table 9

Table 1 0

Table 11

Table 1 2

Table 1 3

Table 1 4

Table 1 5

Table 1 6

Table 1 7

Table 1 8

Table 1 9

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34 Table 35

Table 36

Table 37

LIST OF TABLES

Transferrin genes sequenced to date.

Sequence alignment of the N-tenninus of human lactoferrin

Summary of the iron binding properties of the mutants.

Crystallisation conditions for different lactoferrin species.

Summary of the conditions used to obtain crystals of FeLfN.

Data collection parameters.

CAD4 Data collection statistics.

Summary of the CAD4 data processing statistics.

Molecular Replacement search models for FeLfN.

Rotation function solutions for FeLfN.

Summary of P1 rigidbody refinement.

Summary of P1 XYZ refmement (Round one).

Summary of P1 XYZ refmement (Round two).

Image plate data collection statistics.

Data processing statistics for the FeLfN image plates.

Translation function results for space group C2. Progress of FeLfN refmement in space group C2 .

Summary of the model used for refmement in space group C2. Comparison of the average B values for FeLfN and Fe2Lf. Summary of the a-helices and J3-sheets in FeLfN.

Summary of the nns deviation between FeLfN and Fe2Lf.

Geometry of the iron site.

Anion/protein hydrogen bonds.

Comparison of the mainchain torsion angles for residues 315 to 326 .

Changes in solvent accessibility.

Summary of the interactions between symmetry related molecules.

Analysis of the contacts made by the common waters.

Internal water molecules.

Setting parameters used for collection of the ApoLfN data in Japan.

Data collection arid processing statistics for the ApoLfN images.

Molecular Replacement search models for ApoLfN. Rotation function solutions for ApoLfN.

Summary of the translation function results.

Summary of ApoLfN rigid body refinement.

Progress of ApoUN refmement.

Summary of the model used for refmement of ApoLfN.

Comparison of the deviations between the three closed N-lobe

structures.

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, Fig. 14

� Fig. 15

� Fig. 16 f � Fig. 17

l Fig. 18

Fig. 19

: Fig. 20

Fig. 2 1

Fig. 22

Fig. 23

Fig. 24

-�'jg. 25

Fig. 26

Fig. 27

Fig. 28

Fig. 29 Fig. 30

Fig. 3 1

Fig. 32

F�g. 33

LIST OF FIGURES

Page

Ribbon structure of iron saturated lactoferrin. 13

Stylised representation of the iron binding site in lactoferrin. 14

Model of the mechanism of iron binding and release. 16

Comparison of LfN30 and a fragment representing the entire N-lobe. 20

Partial restriction map of pUC:hLf. 42

Restriction map of pUC:hLf1 .3 . 43

Sequence of JT30 and JT1 1 . 43

Analysis of the PCR products by agarose gel electrophoresis. 44 Restriction analysis of the 1 .1 kb PCR product. 45

Restriction enzyme analysis of pUC:LfNl. 46

Sequence analysis of the 1.1 kb fragment.

Restriction map of pNUT.

Possible orientations of the LfN cON A in pNUT.

Restriction analysis of pNUT:LfN(for)1 and pNUT:LfN(rev)l.

PCR analysis of genomic DNA isolated from BHK cells.

Analysis of the RNA isolated from BHK cells.

Sequence of the 3 ' primer used to construct the second LfN cDNA

Partial restriction map of pGEM:hLf3.

Strategy used for the construction of the LfN cDNA (version 2!).

Strategy used for the construction of LfN cON A (version 3 !).

Restriction analysis of pGEM:LfN2.

Analysis of the RNA isolated from BHK cells.

Analysis of cell culture media

Purification of LfN by ion exchange chromatography.

Comparison of the UV -visible spectra obtained from iron saturated

LfN and hLf.

Deglycosylation of LfN.

Identification of the lower 37 kDa band found in preparations of LfN

Analysis of the variation in protein purity between different batches.

ESR spectra of iron saturated LfN and hLf.

Comparison of the copper UV -visible spectra obtained from LfN and

hLf

ESR spectra of copper saturated LfN and hLf.

Analysis of the pH dependent release of iron from LfN.

Titration of LfN with �n at pH 7 .8.

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:t: .'

Fig. 49

Fig. 50

Fig. 51

, Fig. 52

Fig. 53

Fig. 54

Fig. 5 5

Fig. 56

Fig. 57

Fig. 58

Fig. 59

Fig. 60

Fig. 61

Fig. 62

Fig. 6 3

Fig. 64

Environment of aspartic acid 60 in Fe2Lf.

Environment of arginine 121 in Fe2Lf. ,

Partial restriction map of pTZ:LfN.

Analysis of the reaction products from a mutagenesis experiment

Summary of the sequencing strategy used to sequence the mutants.

Sequence analysis of mutated clones.

Comparison of. the spectrum of N137A and LfN (400 run to 500 run). I

Analysis of the' N137 NN478A mutant by PAGE and

immunodetection.

Comparison of the spectral properties of D60 S and LfN. Change in R12 1S Amax with decreasing pH.

Comparison of the iron binding properties of LfN and R121S.

ESR spectra o� iron saturated R12 1 D. '

Crystals of DG FeLfN.

Crystals of DG ApoLfN.

Representation of the relationship between the space groups P 1 , I2 andC2

Plot of resolution vs % of observed data with I greater than the

specified sigma levels.

Examples of the fit of the atoms to the density.

Luzzati plot for the refined FeLfN structure.

Plot of the mainchain real space correlation coefficients for FeLfN.

Analysis of the mainchain B values for the refmed FeLfN structure.

Comparison of the density and interactions for Lys 18 and Lys 1 00.

Ramachandran plot for residues 4 to 327 in FeLfN

FeLfN : ribbon and schematic diagrams.

Stereoview of a Ca plot of FeLfN.

Conformation of residues 316 to 327 in Fe2Lf and FeLfN.

Comparison of FeLfN and Fe2Lf after superposition using only

domain 2 .

Th e anion binding site: electron density and contacts.

Superposition of the FeLfN and Fe2Lf iron binding sites.

Schematic diagram of the hydrogen bonding network in and around

the metal and anion binding sites of LfN.

Residues 320 - 326 ; atoms and density.

Hydrogen bonding pattern involving �-strand 1.

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, Fig. 66

'fig. 67

,�g. 68 1f.ig. 69 f Fig. 70

Fig. 71

1Fig. 72

l�ig. 73

,fig. 74

Fig. 75 'Fig. 76

Fig. 77

I

'•

Helix 5: contacts due to the absence of Tyr 324 and presence of Phe

325.

Position of the common and unique waters in FeLfN.

Position and electron density for some waters in the interdomain cleft.

Ribbon diagram of ApoU

Analysis of the ApoLfN data set collected in Japan.

Luzzati plot for the partially refined ApoLfN structure.

Mainchain real. space correlation coefficients for ApoLfN.

Plot of average mainchain B values against residue number.

Ramachandran,plot of mainchain phi/psi angles for ApoLfN.

Cartoon diagram of ApoLfN

Comparison of the crystal packing in ApoLfN and FeLfN.

ApoLfN metal binding site showing the unassigned electron density.

Comparison of the position of the iron binding ligands in ApoLfN

and FeLfN.

V1l1

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