Exploring Amino Acids and Proteins: Structure and Reactivity

1. Amino acids, Peptides, Proteins Andy Howard Introductory Biochemistry10 September 2014, IIT

2. Proteins are worth studying • We’ll finish our explanation of amino acid properties, and then move on to peptide and protein structures Amino Acids, Peptides, Proteins

3. Amino acids, concluded Peptides, proteins The notion of protein structure Levels of protein structure Domains TIM barrels Generalizations about structure Plans Amino Acids, Peptides, Proteins

4. iClicker question #1 1. What amino acids are in ELVIS? • (a) asp - lys - val - ile - ser • (b) asn - lys - val - ile - ser • (c) glu - leu - val - ile - ser • (d) glu - lys - val - ile - ser • (e) Thank you very much. (25 seconds) Amino Acids, Peptides, Proteins

5. iClicker question #2 • 2. How many of the twenty plentiful, ribosomally encoded amino acids have exactly one chiral center? • (a) zero • (b) one • (c) seventeen • (d) eighteen • (e) twenty Amino Acids, Peptides, Proteins

6. 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, Peptides, Proteins

7. 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, Peptides, Proteins

8. 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, Peptides, Proteins

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

10. Don’t fall into the trap! • Ionization of leucine: Amino Acids, Peptides, Proteins

11. 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, Peptides, Proteins

12. 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 N: • Lys sidechain –NH3+pKa = 10.5 • Arg sidechain =NH2+pKa = 12.5 Amino Acids, Peptides, Proteins

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

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

15. 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, Peptides, Proteins

16. Resonance-stabilized ion Amino Acids, Peptides, Proteins

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

18. Phosphorylation • ATP donates terminal phosphate to side-chain hydroxyl of ser, thr, tyr • ATP + Ser-OH  ADP + Ser-O-(P) • Often involved in activating or inactivating enzymes • Under careful control of enzymes called kinases and phosphatases • This is an instance of post-translational modification Amino Acids, Peptides, Proteins

19. Amino acid frequencies and importance in active sites • Polaramino acids, particularly S, H, D, T, E, K, are at the heart of most active sites of enzymes and other globular proteins • Yet they’re relatively uncommon overall in proteins • Nonpolar amino acids (V, L, I, A) occur with higher frequencies overall Amino Acids, Peptides, Proteins

20. 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, Peptides, Proteins

21. Growth of oligo- or polypeptide Amino Acids, Peptides, Proteins

22. 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, Peptides, Proteins

23. Double-bond character of peptide Amino Acids, Peptides, Proteins

24. The result: planarity! • This partial double bond character means the amide nitrogen (like the carbonyl carbon) 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, Peptides, Proteins

25. Rotations and flexibility • Planarity implies  = 180º, where  is the torsion 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, Peptides, Proteins

26. Ramachandran angles G.N. Ramachandran Amino Acids, Peptides, Proteins

27. 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, Peptides, Proteins

28. 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 (< 5º) from 180º. • Aromatic amino acids (F, Y, W) are the most likely to depart from planarity Amino Acids, Peptides, Proteins

29. 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, Peptides, Proteins

30. Cis- vs. trans- peptides • Determined by positions of successive alpha carbons • If 2 successive alpha carbons are on opposite sides of the peptide bond, it’s trans; if they’re on the same side, it’s cis Fig. courtesy of Voet & Voet, Biochemistry Amino Acids, Peptides, Proteins

31. Why trans peptides are more common • Trans is much more common because the side chains are less likely to overlap • Exception: amide nitrogen of proline—cis is only a little more interfering than trans Figure courtesy of Wikimedia Amino Acids, Peptides, Proteins

32. 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, Peptides, Proteins

33. 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, Peptides, Proteins

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

35. iClicker question 3. What’s the net charge on ELVIS at pH 7? • (a) 0 • (b) +1 • (c) -1 • (d) +2 • (e) -2 Amino Acids, Peptides, Proteins

36. iClicker question 4 4. An amino acid within a protein has main-chain torsion angles =-90, =-60. This amino acid is probably • (a) part of a right-handed -helix • (b) part of a -sheet • (c) part of a left-handed -helix • (d) outside of any secondary structure • (e) none of the above Amino Acids, Peptides, Proteins

37. 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, Peptides, Proteins

38. 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, Peptides, Proteins

39. What does it mean to characterize a molecule’s structure? • In general, covalent bonds are of constant length and produce constant bond angles • Single covalent bonds, though, allow for rotation about the bond • Therefore the 3-D structure can either be floppy or well-defined • Peptides and noncyclic sugars flop (mostly) • Proteins: mostly well-defined;a little flexibility on the surface Amino Acids, Peptides, Proteins

40. Proteins have well-defined structures • This is not necessarily obvious! • Many biopolymers, notably polysaccharides and to some degree polynucleotides, are floppier • Proteins do have some flexibility, particularly near their surfaces, but they behave approximately like rigid bodies under many circumstances. 1E7H Amino Acids, Peptides, Proteins

41. Protein Structure Helps us Understand Protein Function • If we do know what a protein does, its structure will tell us how it does it. • If we don’t know what a protein does, its structure might give us what we need to know to figure out its function. Amino Acids, Peptides, Proteins

42. Levels of Protein Structure:G&G §5.1 • We conventionally describe proteins at four levels of structure, from most local to most global: • Primary: linear sequence of peptide units and covalent disulfide bonds • Secondary: main-chain H-bonds that define short-range order in structure • Tertiary: three-dimensional fold of a polypeptide • Quaternary: Folds of multiple polypeptide chains to form a complete oligomeric unit Amino Acids, Peptides, Proteins

43. What does the primary structure look like? (G&G §5.3) • -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, Peptides, Proteins

44. Components of secondary structure (G&G §6.3) • , 310,  helices • pleated sheets and the strands that comprise them • Beta turns • More specialized structures like collagen helices Amino Acids, Peptides, Proteins

45. An accounting for secondary structure: phospholipase A2 Amino Acids, Peptides, Proteins

46. Alpha helix (G&G fig. 6.6) Amino Acids, Peptides, Proteins

47. Characteristics of  helices(G&G Fig. 6.9) • 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, for the most part • ~ 10 residues long in globular proteins Amino Acids, Peptides, Proteins

48. 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, Peptides, Proteins

49. 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, Peptides, Proteins

50. 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, Peptides, Proteins