Frederick SANGER (1918-)
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Transcript of Frederick SANGER (1918-)
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:[email protected] instructor’s e-mail address