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Frederick SANGER (1918-)

1958 Nobel Prize in Chemistry: for his work on the structure of proteins, especially that of insulin. GIVEQCCASVCSLYQLENYCN PVNQHLCGSHLVEALYLVCGERGFFYTPKA. Frederick SANGER (1918-).

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Frederick SANGER (1918-)

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  1. 1958 Nobel Prize in Chemistry:for his work on the structure of proteins,especially that of insulin GIVEQCCASVCSLYQLENYCNPVNQHLCGSHLVEALYLVCGERGFFYTPKA Frederick SANGER(1918-) 1980 Nobel Prize in Chemistry (1/4):for his contribution concerning thedetermination of base sequencesin nucleic acids

  2. Protein sequencing • Edman degradationup 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

  3. Edman degradation of oligopeptidesTheory of automatic sequencing

  4. Liquid chromatogram of the PTH derivatives of 20 amino acids Derivatives ofdifferent aminoacids can be distinguished AND identified bytheir elution times.

  5. 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 ofstep 1: 100% R1 100% R1step 2: 95% R2 + 5% R1 99% R2 + 1% R1step 10: 63% R10 + 30% R9 91% R10 + 8% R9step 20: 38% R20 + 38% R19 + 18% R18 83% R20 + 16% R19step 50: 8% R50 + 21% R49 + 26% R48 61% R50 + 30% R49 + 7% R48

  6. Irreversible cleavage of cystine bridges

  7. Recombinant DNA technologythe human genome is “known”and the DNAamino acid dictionary is known, too However,the encoded, the nascent and the nativesequences are NOT necessarily the same

  8. known some uncertainty some uncertainty known some uncertainty …and this is what we wouldlike to figure out...

  9. Nobel Prize inChemistry2002(1/4 - 1/4) John B. FENN Koichi TANAKA for their development of soft desorption ionisation methods formass spectrometric analyses of biological macromolecules

  10. MALDI-TOF MS: Matrix AssistedLaser DesorptionIonization - Time Of FlightMass Spectrometer

  11. ESI MS: ElectroSpray Ionization Mass Spectrometer

  12. MS-MS or Tandem Mass Spectrometry

  13. 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

  14. Robert Bruce MERRIFIELD1984, 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

  15. 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

  16. 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 

  17. Due to sterichindrance=0 AND =0cannot occur.Only certain pairsof values are permitted by thegeometries of thesuccessive peptidebonds

  18. Ramachandran - plot for Ala

  19. Typical occurrences of each amino acids in secondary structures

  20. Ramachandran plot of pyruvate kinase (except Gly’s)

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

  22. X-ray diffraction X-rays are scattered byelectrons around nucleiScattering pattern can beused for calculation of the positions of nucleiThe sample is asingle crystal

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

  24. Preparation of single crystals In most cases it is by far not easy as it seems...

  25. Nuclear Magnetic Resonance (NMR)

  26. 1H NMR spectrum of lysozyme

  27. 1 mM [13C-15N] Ubiquitin in 90% H2O-10% D2O13C-1H HSQC - An example for interaction through a chemical bond

  28. 1 mM [13C-15N] Ubiquitin in 90% H2O-10% D2OHNCA - An example for interaction through multiple bonds

  29. Nuclear Overhauser Effect (NOE) - Interaction through the space

  30. 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) X-ray vs NMR

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