CH339K

1. CH339K Proteins: Amino Acids, Primary Structure, and Molecular Evolution

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…

8. 2 Amides

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

13. Ionizable Side Chains

14. Hydrogen Bond Donors / Acceptors

15. Disulfide formation

16. Modified Amino Acids

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

18. 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

19. Corynebacteriumdiphtheriae Corynebacteriophage

20. 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

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

22. Other Amino Acids

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

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

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

30. 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)

31. Peptide Bond

32. Resonance stabilization of peptide bond

33. 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

40. Molecular Evolution

41. Sequence differences among vertebrate hemoglobins Time of Divergence |-------------|-------------|------------|------------|-------------|------------| ┌───────────────────────────────Shark │ │┌─────────────────────Perch └─────────┤ │┌─────────────Alligator └───────┤ │┌──────Horse └──────┤ │┌───Chimp └──┤ │ └───Human |-------------|-------------|------------|------------|------------|------------|------------|------------| Sequence Difference

42. Neutral Theory of Molecular Evolution • Kimura (1968) • Mutations can be: • Advantageous • Detrimental • Neutral (no good or bad phenotypic effect) • Advantageous mutations are rapidly fixed, but really rare • Diadvantageous mutations are rapidly eliminated • Neutral mutations accumulate

43. What Happens to a Neutral Mutation? • Frequency subject to random chance • Will carrier of gene reproduce? • Many born but few survive • Partly selection • Mostly dumb luck • Gene can have two fates • Elimination (frequent • Fixation (rare)

44. Genetic Drift in Action Our green genes are evolutionarily superior! Never mind… Ow!

45. Simulation of Genetic Drift • 100 Mutations x 100 generations: • 1 gets fixed • 2 still exist • 97 eliminated (most almost immediately)

46. Rates of Change

47. Protein Evolution RatesDifferent proteins have different rates

48. Protein Evolution RatesDifferent proteins have different rates

49. Rates (cont.) • Slow rates in proteins critical to basic functions • E.g. histones ≈ 6 x 10-12 changes/a.a./year

50. Rates(cont.) • Fibrinopeptides • Theoretical max mutation rate • Last step in blood clotting pathway • Thrombin converts fibrinogen to fibrin