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

2.  -Amino Acid 

3. All amino acids as incorporated are in the L-form All amino acids as incorporated are in the L-form Some amino acids can be changed to D- after incorporation Some amino acids can be changed to D- after incorporation D-amino acids occur in some non-protein molecules 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. 4-HydroxyprolineCollagen 5-HydroxylysineCollagen 6-N-MethyllysineHistones  -Carboxygultamate Clotting factors DesmosineElastin SelenocysteineSeveral enzymes (e.g. glutathione peroxidase) Modified Amino Acids

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

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

19. Corynebacterium diphtheriae 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 adds a bulky group to diphthamide eEF2 is inactivated Cell quits making protein Cell(s) die Victim dies Diphtheria Toxin Action

22. Other Amino Acids

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

28. Polymerization  G 0 ’ = +10-15 kJ/mol  G 0 ’ = +10-15 kJ/mol

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

30. In vitro, a starting amino acid can be coupled to a solid matrix In vitro, a starting amino acid can be coupled to a solid matrix Another amino acid with Another amino acid with A protected amino group A protected amino group An activating group at the carboxy group An activating group at the carboxy group Can be coupled Can be coupled This method runs backwards from in vivo synthesis (C  N) 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 Other amino acids have a trans-cis ratio of ~ 1000:1 Prolines have cis:trans ratio of ~ 3:1 Prolines have cis:trans ratio of ~ 3:1 Ring structure of proline minimizes  G 0 difference Ring structure of proline minimizes  G 0 difference

40. MOLECULAR EVOLUTION

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

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 Ow! Our green genes are evolutionarily superior! Never mind…

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 Rates Different 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

51. Fibrinopeptides keep fibrinogens from sticking together.

52. Rates (cont.) Only constraint on sequence is that it has to physically be there Fibrinopeptide limit ≈ 9 x 10 -9 changes/a.a./year

53. Amino acid sequences of several ribosome-inhibiting proteins

54. Phylogenetic trees built from the amino acid sequences of type 1 RIP or A chains (A) and B chains (B) of type 2 RIP (ricin-A, ricin-B, and lectin RCA- A and RCA-B from castor bean; abrin-A, abrina/b-B, and agglutinin APA-A and APA-B from A. precatorius; SNAI-A and SNAI-B, SNAV-A and SNAV-B, SNAI'-A and SNAI'-B, LRPSN1-A and LRPSN1-B, LRPSN2-A and LRPSN2-B, and SNA- IV from S. nigra; sieboldinb-A, sieboldinb-B, SSAI-A, and SSAI-B from S. sieboldiana; momordin and momorcharin from Momordica charantia; MIRJA from Mirabilis jalapa; PMRIPm-A and PMRIPm-B, PMRIPt-A and PMRIPt-B from Polygonatum multiflorum; RIPIriHol.A1, RIPIriHol.A2, and RIPIriHol.A3 from iris hybrid; IRAr-A and IRAr-B, IRAb-A and IRAb-B from iris hybrid; SAPOF from S. officinalis; luffin-A and luffin-B from Luffa cylindrica; and karasurin and trichosanthin from Trichosanthes kirilowii) Hao Q. et.al. Plant Physiol. 2010:125:866-876

55. Phylogenetic tree of Opisthokonts, based on nuclear protein sequences Iñaki Ruiz-Trillo, Andrew J. Roger, Gertraud Burger, Michael W. Gray & B. Franz Lang (2008) Molecular Biology and Evolution, Jan 9