Protein Basics

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 • Enzymes • Catalyze biological reactions • Structural role • Cell wall • Cell membrane • Cytoplasm

3. Protein Structure

4. 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 NH2-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 (Cα) attached to: • Hydrogen (H) • Amino group (-NH2) • Carboxyl group (-COOH) • Side chain (R)

12. General Amino Acid Structure H H2N COOH Cα R

13. General Amino Acid Structure

14. Amino Acids • Chiral

15. Chirality: Glyceraldehyde D-glyderaldehyde L-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 • τABCD 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 • τABCD 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. Backbone Torsion Angles • Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2

35. Backbone Torsion Angles

36. Backbone Torsion Angles • Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2 • Dihedral angle φ : rotation about the bond between N and Cα

37. Backbone Torsion Angles

38. Backbone Torsion Angles • Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2 • Dihedral angle φ : rotation about the bond between N and Cα • Dihedral angle ψ : rotation about the bond between Cα and the carbonyl carbon

39. Backbone Torsion Angles

40. 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

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. 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 • 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. 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 cytochrome C-256 Ramachandran plot alpha-helix

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