Amino Acids & Proteins

1. Amino Acids & Proteins

2. Amino Acids • Amino acid: A compound that contains both an amino group and a carboxyl group. • -Amino acid: An amino acid in which the amino group is on the carbon adjacent to the carboxyl group. • although -amino acids are commonly written in the unionized form, they are more properly written in the zwitterion (internal salt) form.

3. Chirality of Amino Acids • With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the -carbon) and are chiral. • the vast majority have the L-configuration at their -carbon.

4. Amino Acids with Nonpolar side chains

5. With Polar side chains

6. With Acidic & Basic Side Chains

7. Acid-Base properties, Non polar and polar side chains

8. Acid-Base Properties, Acidic/Basic Side Chains

9. Acidity: -COOH Groups • The average pKa of an -carboxyl group is 2.19, which makes them considerablystrongeracidsthan acetic acid (pKa 4.76). • The greater acidity is accounted for by the electron-withdrawing inductive effect of the adjacent -NH3+ group. Note that the NH2 will be protonated at these low pHs

10. Acidity: side chain -COOH • Due to the electron-withdrawing inductive effect of the -NH3+ group, side chain -COOH groups are also stronger than acetic acid. • The effect decreases with distance from the -NH3+ group. Compare: -COOH group of alanine (pKa 2.35) -COOH group of aspartic acid (pKa 3.86) -COOH group of glutamic acid (pKa 4.07)

11. Acidity: -NH3+ groups • The average value of pKa for an -NH3+ group is 9.47, compared with a value of 10.76 for a 1° alkylammonium ion.

12. Details: The Guanidine Group of Arg • The basicity of the guanidine group is attributed to the large resonance stabilization of the protonated form relative to the neutral form.

13. Details: Imidazole Group • The imidazole group is a heterocyclic aromatic amine.

14. Useful Recall from buffer solutions Ka = [H+] [A-]/[HA] If Ka = [H+] then [A-] = [HA]

15. Isoelectric Point • Isoelectric point (pI): pH at which an amino acid, polypeptide, or protein has a total charge of zero. • The pI for glycine, for example, falls between the pKa values for the carboxyl and amino groups.

16. Isoelectric Point of glycine continued Again pKa = pH-log([conj base]/[acid]) At pH = 6.06 For carboxyl group 2.35 = 6.06 - log ([RCO2-]/[RCO2H]) 6.06- 2.35 = 3.71 = log ([RCO2-]/[RCO2H]) ([RCO2-]/[RCO2H]) = 103.71 or 99.98% ionized as neg ion. For amino group 9.78 = 6.06 - log([RNH2]/[RNH3+]) ([RNH2]/[RNH3+]) = 10-3.72 or 99.98% protonated. On average Zero Chg.

17. Furthermore….. A+ B C- [A+] = [C-]

18. Titration of conjugate acid of glycine with NaOH. Strongly acid -CO2H and -NH3+ Buffer solution. [-CO2H] = [-CO2-] Isoelectric. Zero net charge. Buffer solution. [-NH3+]=[-NH2] Strongly basic -CO2- and NH2

19. Isoelectric Point If pH is lower than pI then more protonated molecules. If higher then more negative charge.

20. Isoelectric Point Three buffered pHs

21. Aspartic acid B A+ C- D2- pKa 3.86 9.82 2.10 Note species B has zero net charge. pKa1 and pKa2 control [A+] and [C-] which should be equal. pH = pKa [A+] = [B] [B] = [C-] pI = (2.10 + 3.86)/2 [A+] = [C-] [D2-] approx 0

22. Arginine A2+ B+ C D- pI = (9.04+12.48)/2 =10.76 [B+] = [D-]; [A2+] about 0

23. Electrophoresis • Electrophoresis: The process of separating compounds on the basis of their electric charge. • electrophoresis of amino acids can be carried out using paper, starch, polyacrylamide and agarose gels, and cellulose acetate as solid supports.

24. Electrophoresis • A sample of amino acids is applied as a spot on the paper strip. • An electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own. • Molecules with a high charge density move faster than those with low charge density. • Molecules at isoelectric point remain at the origin. • After separation is complete, the strip is dried and developed to make the separated amino acids visible. • After derivitization with ninhydrin, 19 of the 20 amino acids give the same purple-colored anion; proline gives an orange-colored compound.

25. Electrophoresis • The reagent commonly used to detect amino acid is ninhydrin.

26. Polypeptides & Proteins • In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds to which he gave the name peptide bonds. • Peptide bond:The special name given to the amide bond between the -carboxyl group of one amino acid and the -amino group of another.

27. Serinylalanine (Ser-Ala)

28. Peptides • Peptide:The name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain. • Dipeptide: A molecule containing two amino acids joined by a peptide bond. • Tripeptide: A molecule containing three amino acids joined by peptide bonds. • Polypeptide: A macromolecule containing many amino acids joined by peptide bonds. • Protein: A biological macromolecule of molecular weight 5000 g/mol or greater, consisting of one or more polypeptide chains.

29. Writing Peptides • By convention, peptides are written from the left, beginning with the free -NH3+ group and ending with the free -COO- group on the right.

30. Writing Peptides • The tetrapeptide Cys-Arg-Met-As • At pH 6.0, its net charge is +1. At pH 8 it would be half ionized.

31. Primary Structure • Primary structure: The sequence of amino acids in a polypeptide chain; read from the N-terminal amino acid to the C-terminal amino acid: • Amino acid analysis: • Hydrolysis of the polypeptide, most commonly carried out using 6M HCl at elevated temperature. • Quantitative analysis of the hydrolysate by ion-exchange chromatography.

32. Ion Exchange Chromatography • Analysis of a mixture of amino acids by ion exchange chromatography

33. Cyanogen Bromide, BrCN • BrCN Selectively cleaves of peptide bonds formed by the carboxyl group of methionine.

34. Cyanogen Bromide, BrCN Mechanism to follow

35. Cyanogen Bromide, BrCN • Step 1: Nucleophilic displacement of bromine.

36. Cyanogen Bromide, BrCN • Step 2: Internal nucleophilic displacement of methyl thiocyanate.

37. Cyanogen Bromide, BrCN • Step 3: Hydrolysis of the imino group.

38. Enzyme Catalysis • A group of protein-cleaving enzymes called proteases can be used to catalyze the hydrolysis of specific peptide bonds.

39. Edman Degradation • Edman degradation: Cleaves the N-terminal amino acid of a polypeptide chain.

40. Edman Degradation • Step 1: Nucleophilic addition to the C=N group of phenylisothiocyanate and proton tautomerization

41. Edman Degradation • Step 2: Nucleophilic addition of sulfur to the C=O of the adjacent amide group and acid catalysis.

42. Edman Degradation • Step 3: Isomerization of the thiazolinone ring. substitution

43. DNFB tagging of N terminal AA

44. Carboxypeptidase cleavage of C terminal AA • Treatment of peptide with carboxypeptidase cleaves the peptide linkage adjacent to the free alpha carboxyl group. It may then be identified.

45. Primary Structure, example Deduce the 1° structure of this pentapeptide Glu- Glu-His-Phe Glu-His-Phe-Arg-Ser using

46. Polypeptide Synthesis • The problem in protein synthesis is how to join the -carboxyl group of aa-1 by an amide bond to the -amino group of aa-2, and not vice versa.

47. Polypeptide Synthesis Strategy • Protect the -amino group of aa-1. • Activate the -carboxyl group of aa-1. • Protect the -carboxyl group of aa-2.

48. Amino-Protection • convert them to amides.

49. Amino-Protection • Treatment of an amino group with either of these reagents gives a carbamate (an ester of the monoamide of carbonic acid). • A carbamate is stable to dilute base but can be removed by treatment with HBr in acetic acid.

50. Amino-Protecting Groups • The benzyloxycarbonyl group is also removed by hydrogenolysis. • The intermediate carbamic acid loses carbon dioxide to give the unprotected amino group.