1. Amino Acids and Proteins Muhammad Jawad Hassan Assistant Professor Biochemistry

2. Objectives Structure and Classification of amino acids Peptide Bond and Primary structure of protein Secondary Structure of protein, Helices and Sheets Tertiary and Quaternary Structure of protein, domain and motifs Structure-function relationship of proteins and disease

3. 3 Structure dictates function Protein structure allows DNA replication without dissociation of replicating machinery

4. Protein subunits:  amino acids: L & D isomers Mirror images of each other R group = side chains Amino group Carboxylic acid group Only L amino acids found in proteins. C  chiral, L & D isomers not symmetrical, except glycine

5. The 20 Amino Acids The amino acids each have their own shape and charge due to their specific R group. View the molecular shape of amino acids by clicking on the URL link below: http://sosnick.uchicago.edu/amino_acids.html Would the shape of a protein be affected if the wrong amino acid were added to a growing protein chain?

6. Ionization state as a function of pH Physiological pH (measure of [H + ])

7. Simplest amino acids Ball & stick Stereochemical Fischer projections

8. Aliphatic side chains M: thioether (-S-) Ile: 2nd chiral center Aliphatic side chains hydrophobic

9. Proline: cyclic structure Ring structure: Proline conformationally restricted, marked effect on protein architecture

10. Aromatic side chains

11. Cysteine Similar to Serine with sulfhydryl, or thiol (-SH) group replacing hydroxyl (-OH) group -SH more reactive than -OH. -SH pairs form disulfide bonds (aka bridges), key role stabilizing proteins

12. The basic amino acids Polar side chains Lys & Arg have positive charges at neutral pH His can be positively charged near physiological pH Lys side chain capped with amino group

13. Carboxylate & Carboxamide side chains

14. pK a of some amino acids

15. Amino acid abbreviations

16. 16 Essential Amino Acids 10 amino acids not synthesized by the body arg, his, ile, leu, lys, met, phe, thr, trp, val Must obtain from the diet All in diary products 1 or more missing in grains and vegetables

17. Primary structure: Peptide bond, between AAs Between  -carboxyl group of one AA &  -amino group of another 2 amino acids Dipeptide Loss of H 2 O Equilibrium favors hydrolysis, hence, biosynthesis of peptide bonds require free energy input Peptide bonds are stable kinetically

18. Polypeptide chain has direction

19. Main chain or backbone Constant backbone: regularly repeating part Distinctive side chains (R-groups): variable part AA unit in a polypeptide is called a residue, which contains, a carbonyl group; good hydrogen-bond acceptor, an NH group (except Pro); good hydrogen-bond donor

20. Cross links (disulfide bridges) Prevalent mainly in extracellular proteins

21. Bovine insulin: AA sequence 1953, Fred Sanger determined aa sequence of insulin, landmark! Showed for 1st time, protein has precisely defined aa sequence Also showed that only L-amino acids were present, linked by peptide bonds Now, aa sequence of > 100,000 proteins are known 1950s-1960s studies showed aa sequence genetically determined Each of 20 aa encoded by one or more specific sequences of 3 nucleotides.

22. Polypeptide bonds are planar Six atoms (C a, C, O, N, H, C a ) lie in a plane, in a pair of aa

23. Bond lengths in peptide unit

24. Trans & cis peptides Cis configuration has steric hindrance; trans strongly favored

25. Rotation of bonds in a polypeptide Amino group to C  & carbonyl group to C  are pure single bonds, allow rotation Freedom of rotation allows proteins to fold in different ways Dihedral angle: measure of rotation about a bond between -180 o & +180 o

26. Ramachandran diagram Most angle combinations (75%) excluded by steric hindrance Dark green most favored Steric exclusion: powerful organizing principle Limited conformations favor protein folding, favorable entropy of too many conformations opposes folding

27. Secondary structure: (1) alpha helix 1951, predicted by Pauling & Corey, 6 years before it was seen! ribbon ball & stick, side end view space-filling core

28. alpha helix stabilized by hydrogen bonds CO group of residue n forms H-bond with NH group of Residue n + 4

29. Ball & stick model of alpha helix

30. Ribbon and cylindrical depiction Residues related to each other by a rise of 1.5 Å and a rotation of 100 degrees. 3.6 aa residues / turn Pitch = 5.4 Å(1.5x3.6)

31. Ferritin, an iron storage protein 75% alpha helix Helical content of proteins ranges widely

32. Super helix: alpha helical coiled coil Can be as long as 1000 Å, very stable Helical cables in these proteins serve a mechanical role, forming stiff bundles of fibers Found in: myosin and tropomyosin in muscle, fibrin in blood clots, keratin in hair, quills, claws, hoofs, & horns intermediate filaments (cytoskeleton or internal scaffolding of cells)

33. Structure of a beta strand Side chains are alternately above and below plane of backbone Distance between adjacent aa = 3.5 A Contrast to 1.5 A for alpha helix Also predicted by Pauling & Corey

34. Antiparallel beta sheet Strands linked by H-bonding between opposite amino acids

35. Parallel beta sheet Strands linked by H-bonding of an aa on one strand to two different aa on the adjacent strand

36. Structure of mixed beta sheet

37. Fatty acid-binding protein Rich in beta sheets Arrow pointing to carboxyl- terminal end

38. Tertiary structure, myoglobin O 2 carrier in muscle, 1st protein in atomic detail, 153 aa, X-ray crystals

39. Tertiary structure, myoglobin, schematic Mainly alpha helices, total = 8 helices (75% of main chain) Prosthetic (helper) group to bind O 2 Heme group is protoporphyrin IX, & central iron atom

40. Distribution of aa in myoglobin Yellow: hydrophobic aa Blue: charged aa White: other aa Cross-section Surface, mainly charged aa. Interior, mainly hydrophobic aa

41. Quaternary structure, dimer Cro protein of bacteriophage lambda Dimer of identical subunits

42. Quaternary structure, tetramer Human hemoglobin, two alpha(red) two beta(yellow) subunits, 4 heme groups Covalent bond…..NO

43. Amino acid sequence determines 3D-structure Bovine ribonuclease, 1950, C. Anfinsen work 4 disulfide bonds 124 amino acids Denature & renature

44. Primary structure determination Acid hydrolysis Column chromatography Ion exchange chromatography

45. Reducing disulfied bonds beta-mercaptoethanol, reduced oxidized

46. Denaturing agent, urea

47. Denaturing agent, guanidinium chloride

48. Denaturing agent, beta mercaptoethanol

49. Ribonuclease: reduction & denaturation

50. Finishing touches: covalent modifications Proteins covalently modified to augment function

51. Research Protein Discuss what you can learn about its structure, function and the organism it comes from using the skills you learned today and website resources. You can explore a number of proteins using Cn3D. Go to the following URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Structure

52. Thank You