Amino Acids, concluded; Proteins and Protein Methods

1. Amino Acids, concluded; Proteins and Protein Methods Andy HowardBiochemistry Lectures, Fall 20101 September 2010 Amino Acids & Proteins

2. Main-chain acid-base chemistry Side-chain reactivity Peptides & Proteins Ramachandran angles Levels of protein structure Primary Secondary Tertiary Quaternary Domains Protein Topology Visualization of structures Plans for Today Amino Acids & Proteins

3. Main-chain acid-base chemistry • Deprotonating the amine group: H3N+-CHR-COO- + OH- H2N-CHR-COO- + H2O • Protonating the carboxylate:H3N+-CHR-COO- + H+H3N+-CHR-COOH • Equilibrium far to the left at neutral pH • First equation has Ka=1 around pH 9 • Second equation has Ka=1 around pH 2 Amino Acids & Proteins

4. Why does pKa depend on the side chain? • Opportunities for hydrogen bonding or other ionic interactions stabilize some charges more than others • More variability in the amino terminus, i.e. the pKa of the carboxylate group doesn’t depend as much on R as the pKa of the amine group Amino Acids & Proteins

5. When do these pKa values apply? • The values given in the table are for the free amino acids • The main-chain pKa values aren’t relevant for internal amino acids in proteins • The side-chain pKa values vary a lot depending on molecular environment:a 9.4 here doesn’t mean a 9.4 in a protein! Amino Acids & Proteins

6. How do we relate pKa to percentage ionization? • Derivable from Henderson-Hasselbalch equation • If pH = pKa, half-ionized: [HA] = [A-] • One unit below: • 91% at more positive charge state, • 9% at less positive charge state • One unit above: 9% / 91% Amino Acids & Proteins

7. Don’t fall into the trap! • Ionization of leucine: Amino Acids & Proteins

8. Side-chain reactivity • Not all the chemical reactivity of amino acids involves the main-chain amino and carboxyl groups • Side chains can participate in reactions: • Acid-base reactions • Other reactions • In proteins and peptides,the side-chain reactivity is more important because the main chain is locked up! Amino Acids & Proteins

9. Acid-base reactivity on side chains • Asp, glu: side-chain COO-: • Asp sidechain pKa = 3.9 • Glu sidechain pKa = 4.1 • That means that at pH = 5.1, a glutamate will be ~90.9% charged • Lys, arg: side-chain nitrogen: • Lys sidechain –NH3+ pKa = 10.5 • Arg sidechain =NH2+ pKa = 12.5 Amino Acids & Proteins

10. Acid-base reactivity in histidine • It’s easy to protonate and deprotonate the imidazole group Amino Acids & Proteins

11. Cysteine: a special case • The sulfur is surprisingly ionizable • Within proteins it often remains unionized even at higher pH Amino Acids & Proteins

12. Ionizing hydroxyls • X–O–H  X–O- + H+ • Tyrosine is easy, ser and thr hard: • Tyr pKa = 10.5 • Ser, Thr pKa = ~13 • Difference due to resonance stabilization of phenolate ion: Amino Acids & Proteins

13. Resonance-stabilized ion Amino Acids & Proteins

14. Other side-chain reactions • Little activity in hydrophobic amino acids other than van der Waals • Sulfurs (especially in cysteines) can be oxidized to disulfides, sulfates, sulfites, … • Nitrogens in his can covalently bond to various ligands • Hydroxyls can form ethers, esters • Salt bridges (e.g. lys - asp) Amino Acids & Proteins

15. Phosphorylation • ATP donates terminal phosphate to side-chain hydroxyl of ser, thr, tyr • Some phosphorylation of H, D, E too • ATP + Ser-OH  ADP + Ser-O-(P) • Often involved in activating or inactivating enzymes • Under careful control of enzymes called kinases and phosphatases Amino Acids & Proteins

16. Peptides and proteins • Peptides are oligomers of amino acids • Proteins are polymers • Dividing line is a little vague:~ 50-80 aa. • All are created, both formally and in practice, by stepwise polymerization • Water eliminated at each step Amino Acids & Proteins

17. Growth of oligo- or polypeptide Amino Acids & Proteins

18. The peptide bond • The amide bond between two successive amino acids is known as a peptide bond • The C-N bond between the first amino acid’s carbonyl carbon and the second amino acid’s amine nitrogen has some double bond character Amino Acids & Proteins

19. Double-bond character of peptide Amino Acids & Proteins

20. The result: planarity! • This partial double bond character means the nitrogen is sp2 hybridized • Six atoms must lie in a single plane: • First amino acid’s alpha carbon • Carbonyl carbon • Carbonyl oxygen • Second amino acid’s amide nitrogen • Amide hydrogen • Second amino acid’s alpha carbon Amino Acids & Proteins

21. Rotations and flexibility • Planarity implies  = 180º, where  is the rotation angle about N-C bond • Free rotations are possible about N-C and C-C bonds • Define  = torsional rotation about N-C • Define  = torsional rotation about C-C • We can characterize main-chain conformations according to ,  Amino Acids & Proteins

22. Ramachandran angles G.N. Ramachandran Amino Acids & Proteins

23. Preferred Values of  and  • Steric hindrance makes some values unlikely • Specific values are characteristic of particular types of secondary structure • Most structures with forbidden values of  and  turn out to be errors Amino Acids & Proteins

24. How far from 180º can w vary? • Remember what we said about the partial double bond character of the C-N main-chain bond • That imposes planarity • In practice it rarely varies by more than a few degrees from 180º. • Aromatic amino acids are the most likely to have non-planar peptides Amino Acids & Proteins

25. Ramachandran plot • Cf. figures in text • If you submit a structure to the PDB with Ramachandran angles far from the yellow regions, be prepared to justify them! Amino Acids & Proteins

26. How to remember f and  • Proteins are synthesized N to C on the ribosome • Therefore the natural way to draw an amino acid is (NH-CHR-CO) • f is the first of those angles •  is the second • f is earlier in the Greek alphabet, and phi comes before psi in Roman spelling Amino Acids & Proteins

27. Why bother with mnemonics? • Few textbooks provide memory aids like these • You’re grown-ups; you can read the actual answers in your textbook • This is intended as a study aid, which is what an instructor should be providing • We’ll do several during the semester Amino Acids & Proteins

28. How are oligo- and polypeptides synthesized? • Formation of the peptide linkages occurs in the ribosome under careful enzymatic control • Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine): • GTP + n-length-peptide + amino acid  GDP + Pi + (n+1)-length peptide Amino Acids & Proteins

29. What happens at the ends? • Usually there’s a free amino end and a free carboxyl end: • H3N+–CHR–CO–(peptide)n–NH–CHR–COO- • Cyclic peptides do occur • Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step. Amino Acids & Proteins

30. Reactivity in peptides & proteins • Main-chain acid-base reactivity unavailable except on the ends • Side-chain reactivity available but with slightly modified pKas. • Terminal main-chain pKavalues modified too • Environment of protein side chain is often hydrophobic, unlike free amino acid side chain Amino Acids & Proteins

31. iClicker question 1 What’s the net charge on ELVIS at pH 7? • (a) 0 • (b) +1 • (c) -1 • (d) +2 • (e) -2 Amino Acids & Proteins

32. iClicker question 2 • Leucine is one of the more common amino acids in proteins. In a typical protein, I would expect the leucine content to be about: • (a) 53% • (b) 7% • (c) 5% • (d) 3% • (e) We do not have enough information to know. Amino Acids & Proteins

33. Disulfides In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains Amino Acids & Proteins

34. What could this do? • Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another • This can influence protein stability Amino Acids & Proteins

35. Proteins have definable structures! • This isn’t intuitively obvious • Many biomolecules are much more conformationally flexible in terms of the number of conformations they actually take on in the real world • Why are protein structures definable? • They’re big enough to have an interior • Hydrophobic in, hydrophilic out imposes order Amino Acids & Proteins

36. Levels of Protein Structure • We conventionally describe proteins at four levels of structure, from most local to most global: • Primary: linear sequence of peptide units • Secondary: main-chain H-bonds that define short-range order in structure • Tertiary: three-dimensional fold of a single polypeptide • Quaternary: Folds of multiple polypeptide chains to form a complete oligomeric unit Amino Acids & Proteins

37. What does the primary structure look like? • -ala-glu-val-thr-asp-pro-gly- … • Can be determined by amino acid sequencing of the protein • Can also be determined by sequencing the gene and then using the codon information to define the protein sequence • Amino acid analysis means percentages; that’s less informative than the sequence Amino Acids & Proteins

38. Components of secondary structure • , 310,  helices • pleated sheets and the strands that comprise them • Beta turns • More specialized structures like collagen helices Amino Acids & Proteins

39. An accounting for secondary structure: phospholipase A2 Amino Acids & Proteins

40. Alpha helix Amino Acids & Proteins

41. Characteristics of  helices • Hydrogen bonding from amino nitrogen to carbonyl oxygen in the residue 4 earlier in the chain • 3.6 residues per turn • Amino acid side chains face outward • ~ 10 residues long in globular proteins Amino Acids & Proteins

42. What would disrupt this? • Not much: the side chains don’t bump into one another • Proline residue will disrupt it: • Main-chain N can’t H-bond • The ring forces a kink • Glycines sometimes disrupt because they tend to be flexible Amino Acids & Proteins

43. Other helices • NH to C=O four residues earlier is not the only pattern found in proteins • 310 helix is NH to C=O three residues earlier • More kinked; 3 residues per turn • Often one H-bond of this kind at N-terminal end of an otherwise -helix •  helix: even rarer: NH to C=O five residues earlier Amino Acids & Proteins

44. Beta strands • Structures containing roughly extended polypeptide strands • Extended conformation stabilized by inter-strand main-chain hydrogen bonds • No defined interval in sequence number between amino acids involved in H-bond Amino Acids & Proteins

45. Sheets: roughly planar • Folds straighten H-bonds • Side-chains roughly perpendicular from sheet plane • Consecutive side chains up, then down • Minimizes intra-chain collisions between bulky side chains Amino Acids & Proteins

46. Anti-parallel beta sheet • Neighboring strands extend in opposite directions • Complementary C=O…N bonds from top to bottom and bottom to top strand • Slightly pleated for optimal H-bond strength Amino Acids & Proteins

47. Parallel Beta Sheet • N-to-C directions are the same for both strands • You need to get from the C-end of one strand to the N-end of the other strand somehow • H-bonds at more of an angle relative to the approximate strand directions • Therefore: more pleated than anti-parallel sheet Amino Acids & Proteins

48. Beta turns • Abrupt change in direction • , angles arecharacteristic of beta • Main-chain H-bonds maintained almost all the way through the turn • Jane Richardson and others have characterized several types Amino Acids & Proteins

49. Collagen triple helix • Three left-handed helical strands interwoven with a specific hydrogen-bonding interaction • Every 3rd residue approaches other strands closely: so they’re glycines Amino Acids & Proteins

50. Reminder re disulfides • Cysteine residues brought into proximity under oxidizing conditions can form a disulfide • Forms a “cystine” residue • Oxygen isn’t always the oxidizing agent • Can bring sequence-distant residues close together and stabilize the protein Amino Acids & Proteins