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CH339K

CH339K. Proteins: Primary Structure, Purification, and Sequencing. a -Amino Acid. a. All amino acids as incorporated are in the L-form Some amino acids can be changed to D- after incorporation D-amino acids occur in some non-protein molecules. I prefer this layout, personally…. 2 Amides.

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CH339K

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  1. CH339K Proteins: Primary Structure, Purification, and Sequencing

  2. a-Amino Acid a

  3. All amino acids as incorporated are in the L-form • Some amino acids can be changed to D- after incorporation • D-amino acids occur in some non-protein molecules

  4. I prefer this layout, personally…

  5. 2 Amides

  6. The Acidic and the Amide Amino Acids Exist as Conjugate Pairs

  7. Ionizable Side Chains

  8. Hydrogen Bond Donors / Acceptors

  9. Disulfide formation

  10. Modified Amino Acids

  11. A Modified Amino Acid That Can Kill You Histidine Diphthamide (2-Amino-3-[2-(3-carbamoyl-3-trimethylammonio-propyl)-3H-imidazol-4-yl]propanoate)

  12. Diphthamide Continued – Elongation Factor 2 • Diphthamide is a modified Histidine residue in Eukaryotic Elongation Factor 2 • EF-2 is required for the translocation step in protein synthesis

  13. Corynebacteriumdiphtheriae Corynebacteriophage

  14. Diphtheria Toxin Action Virus infects bacterium Infected bacxterium produces toxin Toxin binds receptor on cell Receptor-toxin complex is endocytosed Endocytic vessel becomes acidic Receptor releases toxin Toxin escapes endocytic vessel into cytoplasm Bad things happen

  15. Diphtheria Toxin Action • Diphtheria toxin adds a bulky group to diphthamide • eEF2 is inactivated • Cell quits making protein • Cell(s) die • Victim dies

  16. Other Amino Acids

  17. Every a-amino acid has at least 2 pKa’s

  18. Polymerization DG0’ = +10-15 kJ/mol

  19. In vivo, amino acids are activated by coupling to tRNA Polymerization of activated a.a.: DGo’ = -15-20 kJ/mol

  20. In vitro, a starting amino acid can be coupled to a solid matrix • Another amino acid with • A protected amino group • An activating group at the carboxy group • Can be coupled • This method runs backwards from in vivo synthesis (C N)

  21. Peptide Bond

  22. Resonance stabilization of peptide bond

  23. Cis-trans isomerization in prolines • Other amino acids have a trans-cis ratio of ~ 1000:1 • Prolines have cis:trans ratio of ~ 3:1 • Ring structure of proline minimizes DG0 difference

  24. Physical Methodsor How to Purify and Sequence a Weapons-Grade Protein

  25. First Question How do I measure the amount of protein I have?

  26. UV Absorption Spectrophotometry

  27. Beer-Lambert Law c = concentration l = path length e = extinction coefficient An Absorbance = 2 means that only 1% of the incident beam is getting through.

  28. Transmittance and Absorbance Absorbance vs. Concentration Transmittance vs. Concentration

  29. Second Question How can I spot my protein in the great mass of different proteins?

  30. Electrophoresis

  31. The frictional coefficient f depends on the size of the molecule, which in turn depends upon the molecular mass, so: i.e. the velocity depends on the charge/mass ratio, which varies from protein to protein

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