Frederick SANGER(1918-)

1958 Nobel Prize in Chemistry:for his work on the structure of proteins,especially that of insulin

1980 Nobel Prize in Chemistry (1/4):for his contribution concerning thedetermination of base sequencesin nucleic acids

GIVEQCCASVCSLYQLENYCN

PVNQHLCGSHLVEALYLVCGERGFFYTPKA

Protein sequencing• Edman degradation

up to ~20-30 residues, time consuming,needs large amount of purified protein

• Recombinant DNA technologyfast, but it does not consider splicing andposttranslational modifications

• Mass Spectrometrynot for exact sequencing of long chains, rather for cataloging of cellular proteins

Edman degradation of oligopeptidesTheory of automatic sequencing

Liquid chromatogram of the PTH derivatives of 20 amino acids

Derivatives ofdifferent aminoacids can be distinguished AND identified bytheir elution times.

Possible problems with Edman degradation:

- multiple peptide chains: the primary result of sequencing of the native protein consisting ofn peptide chains would be n amino acids for each positionin the sequence it is impossible to decide which amino acid follows which

- imperfection (<100% efficiency): if cleavage is performed with 95% OR 99% efficiency, the result of

step 1: 100% R1 100% R1step 2: 95% R2 + 5% R1 99% R2 + 1% R1

step 10: 63% R10 + 30% R9 91% R10 + 8% R9

step 20: 38% R20 + 38% R19 + 18% R18 83% R20 + 16% R19

step 50: 8% R50 + 21% R49 + 26% R48 61% R50 + 30% R49 + 7% R48

Irreversible cleavage of cystine bridges

• Recombinant DNA technologythe human genome is “known”and the DNAamino acid dictionary is known, too

However,the encoded, the nascent and the nativesequences are NOT necessarily the same

known

some uncertainty

known

some uncertainty

some uncertainty

…and this is what we wouldlike to figure out...

cggacgcagagcacagggcgcggctcccacttgcctccggctcccggaagcgggcggggccacctcgcgtcccgggggctaaaacctgcgagcgaggaccgcgcgccaaggctcctcAGCCGAGCGCCGAGCGGTCGATCGCCGTAGCTCCCGCAGCCTGCGATCTCCAGTCTGTGGCTCCTACCAGCCATTGTgtaagtg…0.3kb Intron 1…aatgcagAGGCCAATAATCCGTTATGGAGCATGCCTTTACCCCGTTGGAGCCCCTGCTTTCCACTGgtactgtattt…5.9kb M E H A F T P L E P L L S T aIntron 2…ttttctctagGGAATTTGAAGTACTGCCTTGTAATTCTTAATCAGCCTTTG G N L K Y C L V I L N Q P LaGACAACTATTTTCGTCATCTTTGGAACAAAGgtaacc…8.2kb Intron 3…tgaagCT D N Y F R H L W N K AaCTTTTAAGAGCCTGTGCCGATGGAGGTGCCAACCGCTTATATGATATCACCGAAGGAGAG L L R A C A D G G A N R L Y D I T E G EaAGAGAAAGgtatgc…3.4kb Intron 4…tctttcagCTTTTTGCCTGAATTCATCAAT R E S F L P E F I NaGGAGACTTTGATTCTATTAGGCCTGAAGTCAGAGAATACTATGCTACTAAGgtaaattat G D F D S I R P E V R E Y Y A T Kaaa…2.5kb Intron 5…aatacatagGGATGTGAGCTCATTTCAACTCCTGATCAAGAC G C E L I S T P D Q DaCACACTGACTTTACTAAGTGCCTTAAAATGCTCCAAAAGAAGATAGAAGAAAAAGACTTA H T D F T K C L K M L Q K K I E E K D LaAAGgtaatat…2.5kb Intron 6…tgcttagGTTGATGTGATCGTGACACTGGGAGGC K V D V I V T L G GaCTTGCTGGGCGTTTTGACCAGATTATGGCATCTGTGAATACCTTGTTCCAAGCGACTCAC L A G R F D Q I M A S V N T L F Q A T HaATCACTCCTTTTCCAATTATAATAATCCAAGAGGAATCGCTGATCTACCTGCTCCAACCA I T P F P I I I I Q E E S L I Y L L Q Pagtgagtc…4.3kb Intron 7…tctccttcagGGAAAGCACAGGTTGCATGTAGACACT G K H R L H V D TaGGAATGGAGGGTGATTGGTGTGGCCTTATTCCTGTTGGACAGCCTTGTATGCAGGTTACA G M E G D W C G L I P V G Q P C M Q V TaACCACAGGCCTCAAGTGGAACCTCAgtaagta…9.5kb Intron 8…attttcttacAG T T G L K W N L aCAAATGATGTGCTTGCTTTTGGAACATTGGTCAGTACTTCCAATACCTACGACGGGTCTGT N D V L A F G T L V S T S N T Y D G S aGTGTTGTGACTGTGGAAACTGACCACCCACTCCTCTGGACCATGGCCATCAAAAGCTAACG V V T V E T D H P L L W T M A I K Sa *

Nobel Prize inChemistry

2002

(1/4 - 1/4)

John B. FENN Koichi TANAKA

for their development of soft desorption ionisation methods formass spectrometric analyses of biological macromolecules

MALDI-TOF MS:

Matrix AssistedLaser DesorptionIonization -Time Of FlightMass Spectrometer

ESI MS: ElectroSpray Ionization Mass Spectrometer

MS-MS or Tandem Mass Spectrometry

What can amino acid sequence be used for?

• Searching for similarities by comparison to known sequences classification among protein types (FUNCTION)

• Searching for similarities by comparison to other species evolutionary consequences can be drawn

• Searching for internal repeats history of an individual protein

• Searching for signals designating destination or process control fate of the protein between translation and native state

• Sequence data basis for preparing antibodies specific to the protein

• Sequence data reverse genetics:making DNA probes for the genes encoding the proteins

• Sequence data structures of higher order

PP20/hTPK1 95 DQDHTD 100 … 199 GLKWN 203 … 216 STSN 219Mouse TPK1 95 DQDHTD 100 … 199 GLKWN 203 … 216 STSN 219Schizosaccharomyces pombe 427 CQNTTD 432 … 526 GLEWN 530 … 543 SSCN 546Giardia lamblia 137 EQDSTD 142 … 260 GLKWD 264 … 277 SGCN 280Bacillus cereus 50 EKDQTD 55 … 145 GFKYP 149 … 164 CISN 168Bacillus anthracis Ames 95 EKDQTD 100 … 170 GFKYP 174 … 187 CISN 190Fusobacterium vincentii 78 EKDYTD 83 … 166 GFKYN 170 … 185 CISN 189Enterococcus faecalis 74 EKDDTD 79 … 171 RSKYL 175 … 190 YASN 194Clostridium tetani 75 EKDFTD 80 … 168 GAKYP 172 … 189 GVSN 193Brucella melitensis 73 AKDMTD 78 … 167 NAKWP 171 … 186 TVSN 190

Robert Bruce MERRIFIELD

1984, Nobel Prize in Chemistryfor his development ofmethodology forchemical synthesison a solid matrix

1955, VIGNEAUD:Oxytocin: the first syntheticpeptide hormone(“conquering the Himalayas”)

1902, Emil FISCHER:The first artificial peptide bond

What can you use synthetic peptides for?

•Antigens to stimulate the formation of specific antibodies

•Isolation of receptors for hormones and signalling molecules (affinity cromatography)

•Drugs(e.g. hormone analogs)

•Study of these can help define the rules governing the 3D structure of proteins

Peptide bonds are rigid:torsion does not occur around C(O)-NHbonds

Extent of torsion around N-C bond is denoted by dihedral angle whereas that around C-C(O) bond is denoted by dihedral angle

Due to sterichindrance=0 AND =0cannot occur.

Only certain pairsof values are permitted by thegeometries of thesuccessive peptidebonds

Ramachandran - plot for Ala

Typical occurrences of each amino acids in secondary structures

Ramachandran plot of pyruvate kinase (except Gly’s)

Human Serum Albumin would look like this, if...

X-ray diffraction

X-rays are scattered byelectrons around nuclei

Scattering pattern can beused for calculation of the positions of nuclei

The sample is asingle crystal

Signals of interactions over the space help us to turnthe sequence into 3D structure

Preparation of single crystals

In most cases it is by far not easy as it seems...

Nuclear Magnetic Resonance (NMR)

1H NMR spectrum of lysozyme

1 mM [13C-15N] Ubiquitin in 90% H2O-10% D2O13C-1H HSQC - An example for interaction through a chemical bond

1 mM [13C-15N] Ubiquitin in 90% H2O-10% D2OHNCA - An example for interaction through multiple bonds

Nuclear Overhauser Effect (NOE) - Interaction through the space

X-ray vs NMR• Diffraction: interaction with

electron density• Sample: single crystal

• Result: a “sharp” static snapshot with good spatial resolution

• Perutz and Kendrew (1962)

• Resonance: interaction with magnetic moments

• Sample: isotope labelled protein

• Result: a “blurry”dynamic picture of a conformational ensemble”

• Wüthrich (2002) (1/2)

Stability:“enthalpy side”: formation of bonds“entropy side”: rearrangement of solvent “structure”

Chemical bonds participating in stabilizing protein structureand agents used to cleave them:

• disulfide bridges mercaptoethanol, DTT• H-bonds pH extremes• hydrophobic interactions detergents, urea• ionic interactions changing pH or ionic strength

Peptides Proteins

Motif:A distinct folding pattern forelements of secondary structure;also called a fold orsupersecondary structure.

Domain

A distinct structural unit of a polypeptide;they may have sparate functions andthey may fold as independent, compact units

Subunit

Separate polypeptide chains of the same protein

The same short sequence may take different secondary structuresdepending on its broader environment

Prion:(proteinaceous infectious only)

upon dimerization it suffersdramatic conformational changeleading to spongiformdegeneration (CJD)

Reasons and mechanismare not understood

Proteins

• Functions:

• catalysis enzymes• transport and storage myoglobin, hemoglobin• motion actin myosin• defense skin and hair proteins• regulation hormones, exp. factors• fuel e.g. in plant seeds

Proteins

• Functions:

• CATALYSIS ENZYMES• transport and storage myoglobin, hemoglobin• motion actin myosin• defense skin and hair proteins• regulation hormones, exp. factors• fuel e.g. in plant seeds

1 Oxidoreductases (electron, hydride ion, H atom)

2 Transferases (group transfer, e. g. phosphate, -COO, methyl)

3 Hydrolases (functional groups water)

4 Lyases (formation or saturation of double bonds)

5 Isomerases

6 Ligases (C-C, C-S, C-O, C-N bonds, for ATP)

Enzyme Classification

Each enzyme name ends -ase, except...Each enzyme is (basically) protein, except…

The enzyme exerts its activity in the native conformation,and the reaction takes place at the active site

Enzymes do not usually contain protein only:

apoenzyme (protein) + X = holoenzyme

X = cofactor

cofactor = inorganic ion or coenzyme (or both)

The cofactor bound to the peptide chain by covalent bond is calledprosthetic group

Some enzymes with inorganic ions as cofactorsIron(II)-iron(III) cytochrome oxidase, catalase, peroxidase

Iron-sulfur proteins succinate dehydrogenase, aconitase, dinitrogenase

Copper(II) cytochrome oxidase, superoxide dismutase

Zinc(II) alcohol dehydrogenase, superoxide dismutase

Magnesium hexokinase, glc-6-phosphatase, pyruvate kinase

Potassium pyruvate kinase

Molibdenum dinitrogenase

Selenium glutathione peroxidase

Electron, atom or group transfer coenzymesNAD+ hydride ion nicotinic acid (niacin)

FAD+ electron riboflavin (B2)

CoA ac(et)yl group pantothenic acid

TPP aldehyde group thiamine (B1)

pyridoxal phosphate amino group pyridoxine (B6)

coenzyme B12 H atoms, alkyl groups (B12)

ubiquinone electron

tetrahydrofolate one-carbon groups folic acid

Proteins

• Functions:

• catalysis enzymes• TRANSPORT AND STORAGE

MYOGLOBIN, HEMOGLOBIN• motion actin myosin• defense skin and hair proteins• regulation hormones, exp. factors• fuel e.g. in plant seeds

See also the Molecular Tutorial

Proteins

• Functions:

• catalysis enzymes• transport and storage myoglobin, hemoglobin• MOTION ACTIN, MYOSIN• defense skin and hair proteins• regulation hormones, exp. factors• fuel e.g. in plant seeds

Proteins

• Functions:

• catalysis enzymes• transport and storage myoglobin, hemoglobin• motion actin myosin• DEFENSE SKIN AND HAIR PROTEINS• regulation hormones, exp. factors• fuel e.g. in plant seeds

What do hairdressers do when they make a permanent wave?

Collagen

Collagen helices are cross-linked, mostly by hydroxyproline residues,to the formation of which ascorbic acid (vitamin C) is indispensable that is why the lack of vitamin C caused scurvy

Silk fibroin Spider’s net

Proteins

• Functions:

• catalysis enzymes• transport and storage myoglobin, hemoglobin• motion actin myosin• defense skin and hair proteins• regulation hormones, exp. factors• fuel e.g. in plant seeds

Online resources:

http://bcs.whfreeman.com/lehninger (Ch3, Ch4, Ch5)

see esp. Molecular Tutorial “Protein Architecture”

http://bcs.whfreeman.com/biochem5 (Ch3, Ch4)

For online quizzing give:zoltan.berente@aok.pte.huas instructor’s e-mail address