1. Protein Structure

2. Protein Structure I Primary Structure

3. Figure 5-1 Primary Structure Insulin Signal sequenceChain B MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV C Peptide CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG Chain A SLQKRGIVEQCCTSICSLYQLENYCN Bovine: Insulin Human: ProInsulin

4. Figure 5-1 Primary Structure Insulin Signal sequenceChain B MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV C Peptide CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG Chain A SLQKRGIVEQCCTSICSLYQLENYCN Bovine: Insulin Human: ProInsulin

5. Value of Primary Structure Information Primary sequence information is –prerequisite for determining three-dimensional structure –essential in understanding molecular mechanism of action Sequence comparisons among analogous proteins –provide insights into protein function –reveal evolutionary relationships Sequence of proteins whose mutations result in inherited diseases –assist in development of diagnostic tests –assist in development of effective therapies

6. Primary Structure Determination

7. Strategy Purification of protein to homogeneity Prepare protein for sequencing Sequence polypeptide chains Organize completed structure Alternative: Nucleic Acid Sequencing

8. Figure 5-12 Sequencing Strategy Summary

9. Figure 5-12 Sequencing Strategy I

10. Figure 5-12 Sequencing Strategy II

11. Figure 5-12 Sequencing Strategy III

12. Purification of Protein to Homogeneity

13. Prepare Protein for Sequencing End Group Analysis: How many different subunits Cleavage of disulfide bonds Separation and purification of the polypeptide chains Amino acid composition

14. End Group Analysis (How Many Different Subunits?) N-Terminal Identification

15. Sanger’s Reagent

16. Dansyl Chloride

17. End Group Analysis (How Many Different Subunits?) C-Terminal Identification

18. Reduction

19. Hydrazinolysis

20. Cleavage of Disulfide Bonds

21. Oxidative Cleavage

22. Problem (Oxidation of Methionine to Methionine Sulfone)

23. Reduction and Alkylation

24. Problem

25. Solution

26. Separation and Purification of Polypeptide Chains

27. Sequence Polypeptide Chains Specific peptide cleavage reactions Separation and purification of peptide fragments Sequence determination

28. Hydrolysis Polypeptide Amino Acids Hydrolysis

29. Acid Hydrolysis

30. Mechanism

31. Problems Complete destruction of Trp Partial destruction of Ser, Thr, and Tyr Deamination of Asn and Gln

33. Base Hydrolysis (Many Amino Acids Destroyed) (Racemization)

34. Enzymatic Hydrolysis Mild Conditions Many proteases and peptidases Specific and non-specific Problem: contribution of amino acids from hydrolysis of proteases

35. Amino Acid Analysis (Automated) Ion-exchange chromatography High performance liquid chromatography Colorimetric Analysis

36. Specific Peptide Cleavage Reactions

37. Proteolytic Enzymes Cleave peptide bonds Specificity: R 1

38. Table 5-3 Specificity of Endopeptidases

39. Chemical Cleavage (Cyanogen Bromide)

40. Separation and Purification of Peptide Fragments

41. Sequence Determination: -Edman degradation -Mass Spectrometry

42. Edman Degradation I

43. Edman Degradation II

44. Edman Degradation III

45. Figure 5-16a part 1 Electrospray Ionization Mass Spectrometry (ESI)

46. Figure 5-16a part 2 Electrospray Ionization Mass Spectrometry (ESI)

47. Figure 5-16b Electrospray Ionization Mass Spectrometry (ESI)

48. Figure 5-17 Tandem Mass Spectrometry

49. Organize Completed Structure Ordering peptide fragments Assignment of disulfide bond positions Determine position of amides

50. Ordering Peptide Fragments

51. Figure 5-18 Generating Overlapping Fragments

52. Ordering Peptide Fragments

53. Assignment of Disulfide Bond Positions Hydrolyze without breaking disulfides Reduce, alkylate, and identify linked fragments (disulfides)

54. Assignment of Amide Positions Hydrolyze without breaking amides Hydrolyze fragments and measure NH 3 (need fragments having a single Asn or Gln)

55. Protein Evolution Evolution by Natural Selection Mutations

56. Table 5-5 part 1 Cytochrome c All look like this

57. Sequence Comparisons Provide Information on Protein Structure and Function Homologous proteins: evolutionarily related proteins –Invariant residues –Conservative substitutions –Hypervariable positions Neutral drift

58. Figure 5-21 Phylogenetic Trees Depict Evolutionary History

59. Proteins Evolve by the Duplication of Genes or Gene Segments

60. Protein Families Can Arise through Gene Duplication Orthologous proteins: homologous proteins with the same function in different species Paralogous proteins: independently evolving proteins derived by duplication of a gene (globin family) Pseudogenes

61. Figure 5-22 Globin Family

62. Figure 5-23 The Rate of Sequence Divergence Varies