Incidentally, note the functional groups we have met so far: Hydroxyl Amine Amide Carboxyl

1. Incidentally, note the functional groups we have met so far: Hydroxyl Amine Amide Carboxyl Carbonyl Aldehyde Ketone Ester: Carboxylic acid ester Phosphoester And: Glycosidic bonds C=C double bonds (cis and trans)

2. PROTEINS Amino acids (the monomer of proteins)

3. At pH 7, ,most amino acids are zwitterions (charged but electrically neutral)

5. +OH- ( -H+) +H+ 50-50 charged-uncharged at ~ pH2.5 (=the pK) 50-50 charged-uncharged at ~ pH9 (=the pK) Net charge

6. Numbering (lettering) amino acids ε-amino group ε δ γ β Alpha-carboxyl (attached to the α-carbon) Alpha-amino Alpha-carbon

7. Amino acid examples

9. Shown uncharged (as on exams)

11. Amino acids in 3 dimensions • Asymmetric carbon (4 different groups attached) • Stereoisomers • Rotate polarized light • Optical isomers • Non-superimposable • Mirror images • L and D forms From Purves text

12. Mannose

13. Handout 3-3 (Without showing the R-groups) The backbone is monotonous It is the side chains that provide the variety

14. “Polypeptides” vs. “proteins” • Polypeptide = amino acids connected in a linear chain (polymer) • Protein = a polypeptide or several associated polypeptides (discussed later) • Often used synonymously • Peptide (as opposed to polypeptide) is smaller, even 2 AAs (dipeptide)

15. The backbone is monotonous (Without showing the R-groups) It is the side chains that provide the variety

16. Proteins do most of the the jobs in the cell E.g., egg albumin, hemoglobin, keratin, estrogen receptor,immunoglobulins (antibodies), enzymes (e.g., beta-galactosidase) Each is a polymer or assemblage of polymers made up of amino acidsEach particular protein polymer (polypeptide) has a unique sequence of amino acidsEach molecule of a particular protein has the same sequence of amino acids. E.g., met-ala-leu-leu-arg-glu-leu-val- . . . . How is this sequence determined?

17. Primary (1o) Structure = the sequence of the amino acids in the polypeptide chain

18. asp ser Determining the sequence One way: enzyme: Carboxypeptidase: hydrolyzes the peptide bond , identify e.g., …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp-ser …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu

19. AA mixture (-) (+)

20. A paper electrophoresis apparatus

21. Side view AAs applied at lower end

22. After stopping the paper chromatography and staining for the amino acids: “Rf” 0.82 0.69 0.45 0.27 0.11

23. Paper chromatography apparatus

24. Sub-peptides • Treatment of a polypeptide with trypsin • Trypsin is a proteolytic enzyme. • It catalyzes cleavage (hydrolysis) after lysine and arginine residues Polypeptide chain

25. The order of the subpeptides is unknown. The sequence is reconstructed by noting the overlap between differently produced subpeptides Trypsin (lys, arg) (1) Chymotrypsin (trp, tyr, phe) N C (2)

26. Hemoglobin protein Application to sickle cell disease (Ingram, 1960’s) Fingerprinting a protein: analysis of the sub-peptides (without breaking them down to their constituent amino acids) Sub-peptides No further digestion to amino acids; left as sub-peptides

27. Oligopeptides behave as a composite of their constituent amino acids H H H + H H H - H - Net charge = -1: moves toward the anode in paper electrophoreses Fairly hydrophobic (~5/6): expected to move moderately well in paper chromatography Nomenclature: ala-tyr-glu-pro-val-trp or AYEPVW or alanyl-tyrosyl-glutamyl-prolyl-valyl-tryptophan

28. The mixture of all sub-peptides formed Less negatively charged, More hydrophobic Hb In fingerprinting, these spots contain peptides, not amino acids trypsin Negatively charged ---valine--- (sickle) Positively charged More hydrophobic ---glutamate--- (normal) More hydrophilic Negatively charged Positively charged Negatively charged Positively charged

29. Every different polypeptide has a different primary structure (sequence). Every polypeptide will have different arrangement of spots after fingerprinting.

30. 3-dimensional structure of proteins One given purified polypeptide • Molecule #1: N-met-leu-ala-asp-val-val-lys-.... • Molecule #2: N-met-leu-ala-asp-val-val-lys-... • Molecule #3: N-met-leu-ala-asp-val-val-lys-... • Molecule #4: N-met-leu-ala-asp-val-val-lys-... etc .

31. 3-D structure of polypeptides Arrangement 3 at one momentin time? Arrangement 1 at one momentin time? Arrangement 2 at one momentin time? Arrangement 3 at one momentin time? [Rope models here]

34. Got this far

35. Primary structure itself results in some folding constraints: See bottom of handout 3-3

37. 4 atoms in one plane  6 atoms in one plane

42. There’s still plenty of flexibility

43. Secondary structure: the alpha helix Amino acids shown simplified, without side chains and H’s.

44. Alpha helix depictions C = grays N = blue O = red Poly alanine Side chains = -CH3 (lighter gray) H’s not shown

45. Linus Pauling and a model of the alpha helix.1963

46. H-bond AA residue Secondary structure:

47. Beta sheet

48. Beta-sheets Anti-parallel Parallel

49. secondary structure (my definition): structure produced by regular repeated interactions betweenatoms of the backbone.

50. Neither Beta-sheets Alpha-helices Tertiary structure: The overall 3-D structure of a polypeptide. These “ribbon” depictions do not show the side chains, only the backbone