1. Protein Basics Protein function Protein structure –Primary Amino acids Linkage Protein conformation framework –Dihedral angles –Ramachandran plots Sequence similarity and variation

2. Protein Function in Cell 1.Enzymes Catalyze biological reactions 2.Structural role Cell wall Cell membrane Cytoplasm

3. Protein Structure

5. Hemoglobin – Quaternary Structure Two alpha subunits and two beta subunits (141 AA per alpha, 146 AA per beta)

6. Hemoglobin – Tertiary Structure One beta subunit (8 alpha helices)

7. Hemoglobin – Secondary Structure alpha helix

8. Hydrogen Bonding

9. Hemoglobin – Primary Structure NH 2 -Val-His-Leu-Thr-Pro-Glu-Glu- Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp- Gly-Lys-Val-Asn-Val-Asp-Glu-Val- Gly-Gly-Glu-….. beta subunit amino acid sequence

10. Protein Structure - Primary Protein: chain of amino acids joined by peptide bonds

11. Protein Structure - Primary Protein: chain of amino acids joined by peptide bonds Amino Acid –Central carbon (C α ) attached to: Hydrogen (H) Amino group (-NH 2 ) Carboxyl group (-COOH) Side chain (R)

12. General Amino Acid Structure CαCα H R COOHH2NH2N

13. General Amino Acid Structure

14. Amino Acids Chiral

15. Chirality: Glyceraldehyde L-glyderaldehydeD-glyderaldehyde

16. Amino Acids Chiral 20 naturally occuring; distinguishing side chain

17. 20 Naturally-occurring Amino Acids

18. Amino Acids Chiral 20 naturally occuring; distinguishing side chain Classification: Non-polar (hydrophobic) Charged polar Uncharged polar

19. Peptide Bond Joins amino acids

20. Peptide Bond Formation

21. Peptide Chain

22. Peptide Bond Joins amino acids 40% double bond character –Caused by resonance

24. Peptide bond Joins amino acids 40% double bond character –Caused by resonance –Results in shorter bond length

25. Peptide Bond Lengths

26. Peptide bond Joins amino acids 40% double bond character –Caused by resonance –Results in shorter bond length –Double bond disallows rotation

27. Protein Conformation Framework Bond rotation determines protein folding, 3D structure

28. Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ –Measures orientation of four linked atoms in a molecule: A, B, C, D

30. Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ –Measures orientation of four linked atoms in a molecule: A, B, C, D –τ ABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D

31. Ethane Rotation

32. Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ –Measures orientation of four linked atoms in a molecule: A, B, C, D –τ ABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D –Three repeating torsion angles along protein backbone: ω, φ, ψ

33. Backbone Torsion Angles

34. Dihedral angle ω : rotation about the peptide bond, namely C α 1 -{C-N}- C α 2

35. Backbone Torsion Angles

36. Dihedral angle ω : rotation about the peptide bond, namely C α 1 -{C-N}- C α 2 Dihedral angle φ : rotation about the bond between N and C α

37. Backbone Torsion Angles

38. Dihedral angle ω : rotation about the peptide bond, namely C α 1 -{C-N}- C α 2 Dihedral angle φ : rotation about the bond between N and C α Dihedral angle ψ : rotation about the bond between C α and the carbonyl carbon

39. Backbone Torsion Angles

40. ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair

42. Backbone Torsion Angles ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair φ and ψ are flexible, therefore rotation occurs here

43. Backbone Torsion Angles

44. ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair φ and ψ are flexible, therefore rotation occurs here However, φ and ψ of a given amino acid residue are limited due to steric hindrance Only 10% of the area of the {φ, ψ} space is generally observed for proteins First noticed by G.N. Ramachandran

45. G.N. Ramachandran Used computer models of small polypeptides to systematically vary φ and ψ with the objective of finding stable conformations For each conformation, the structure was examined for close contacts between atoms Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii Therefore, φ and ψ angles which cause spheres to collide correspond to sterically disallowed conformations of the polypeptide backbone

46. Ramachandran Plot Plot of φ vs. ψ Repeating values of φ and ψ along the chain result in regular structure For example, repeating values of φ ~ -57° and ψ ~ -47° give a right-handed helical fold (the alpha- helix) The structure of cytochrome C-256 shows many segments of helix and the Ramachandran plot shows a tight grouping of φ, ψ angles near -50, -50

47. alpha-helix cytochrome C-256 Ramachandran plot

49. Ramachandran Plot White = sterically disallowed conformations (atoms in the polypeptide come closer than the sum of their van der Waals radii) Red = sterically allowed regions (namely right-handed alpha helix and beta sheet) Yellow = sterically allowed if shorter radii are used (i.e. atoms allowed closer together; brings out left-handed helix)

50. Alanine Ramachandran Plot

51. Arginine Ramachandran Plot

52. Glutamine Ramachandran Plot

53. Glycine Ramachandran Plot Note more allowed regions due to less steric hindrance

54. Proline Ramachandran Plot Note less allowed regions due to structure

55. Sequence Similarity Sequence similarity implies structural, functional, and evolutionary commonality Small mutations generally well-tolerated by native structure

56. Sequence Similarity Exception Sickle-cell anemia resulting from one residue change Replace highly polar (hydrophilic) glutamate in hemoglobin with nonpolar (hydrophobic) valine

57. Sickle-cell mutation in hemoglobin sequence

58. Sequence Similarity Exception Sickle-cell anemia resulting from one residue change Replace highly polar (hydrophilic) glutamate in hemoglobin with nonpolar (hydrophobic) valine Causes hemoglobin molecules to repel water and be attracted to one another Leads to the formation of long protein filaments that distort the shape of red blood cells giving them their “sickled” shape Rigid structure of sickle cells blocks capillaries and prevents red blood cells from delivering oxygen