1. 27-1 William H. Brown Beloit College William H. Brown Christopher S. Foote Brent L. Iverson Eric Anslyn http://academic.cengage.com/chemistry/brown Chapter 27 Amino Acids and Proteins

2. 27-2 Amino Acids  Amino acid:  Amino acid: A compound that contains both an amino group and a carboxyl group.  -Amino acid:  -Amino acid: An amino acid in which the amino group is on the carbon adjacent to the carboxyl group. zwitterionalthough  -amino acids are commonly written in the unionized form, they are more properly written in the zwitterion (internal salt) form.

3. 27-3 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. 27-4 Nonpolar side chains

5. 27-5 Polar side chains

6. 27-6 Acidic & Basic Side Chains

7. 27-7 Some Other Amino Acids

8. 27-8 Acid-Base properties

9. 27-9 Acid-Base Properties

10. 27-10 Acidity:  -COOH Groups  The average pK a of an  -carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pK a 4.76). The greater acidity is accounted for by the electron- withdrawing inductive effect of the adjacent -NH 3 + group.

11. 27-11 Acidity: side chain -COOH  Due to the electron-withdrawing inductive effect of the  -NH 3 + group, side chain -COOH groups are also stronger than acetic acid. The effect decreases with distance from the  -NH 3 + group. Compare: pK a 2.35  -COOH group of alanine (pK a 2.35) pK a 3.86  -COOH group of aspartic acid (pK a 3.86) pK a 4.07  -COOH group of glutamic acid (pK a 4.07)

12. 27-12 Acidity:  -NH 3 + groups  The average value of pK a for an  -NH 3 + group is 9.47, compared with a value of 10.76 for a 1° alkylammonium ion.

13. 27-13 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.

14. 27-14 Basicity- Imidazole Group The imidazole group is a heterocyclic aromatic amine.

15. 27-15 Titration of Amino Acids  Titration of glycine with NaOH.

16. 27-16 Isoelectric Point  Isoelectric point (pI):  Isoelectric point (pI): The pH at which an amino acid, polypeptide, or protein has a total charge of zero. The pH for glycine, for example, falls between the pK a values for the carboxyl and amino groups.

17. 27-17 Isoelectric Point

18. 27-18

19. 27-19 Electrophoresis  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.

20. 27-20 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 their 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.

21. 27-21 Electrophoresis The reagent commonly used to detect amino acid is ninhydrin.

22. 27-22 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:  Peptide bond: The special name given to the amide bond between the  -carboxyl group of one amino acid and the  -amino group of another.

23. 27-23 Serinylalanine (Ser-Ala)

24. 27-24 Peptides Peptide: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:Dipeptide: A molecule containing two amino acids joined by a peptide bond. TripeptideTripeptide: A molecule containing three amino acids joined by peptide bonds. PolypeptidePolypeptide: A macromolecule containing many amino acids joined by peptide bonds. ProteinProtein: A biological macromolecule of molecular weight 5000 g/mol or greater, consisting of one or more polypeptide chains.

25. 27-25 Writing Peptides By convention, peptides are written from the left, beginning with the free -NH 3 + group and ending with the free -COO - group on the right.

26. 27-26 Writing Peptides The tetrapeptide Cys-Arg-Met-As At pH 6.0, its net charge is +1.

27. 27-27 Primary Structure  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.

28. 27-28 Ion Exchange Chromatography  Analysis of a mixture of amino acids by ion exchange chromatography

29. 27-29 Cyanogen Bromide, BrCN BrCN Selectively cleaves of peptide bonds formed by the carboxyl group of methionine.

30. 27-30 Cyanogen Bromide, BrCN

31. 27-31 Step 1: Nucleophilic displacement of bromine.

32. 27-32 Cyanogen Bromide, BrCN Step 2: Internal nucleophilic displacement of methyl thiocyanate.

33. 27-33 Cyanogen Bromide, BrCN Step 3: Hydrolysis of the imino group.

34. 27-34 Enzyme Catalysis  A group of protein-cleaving enzymes called proteases can be used to catalyze the hydrolysis of specific peptide bonds.

35. 27-35 Edman Degradation  Edman degradation:  Edman degradation: Cleaves the N-terminal amino acid of a polypeptide chain.

36. 27-36 Edman Degradation Step 1: Nucleophilic addition to the C=N group of phenylisothiocyanate and proton tautomerization

37. 27-37 Edman Degradation Step 2: Nucleophilic addition of sulfur to the C=O of the adjacent amide group with acid catalysis.

38. 27-38 Edman Degradation Step 3: Isomerization of the thiazolinone ring.

39. 27-39 Primary Structure Example 27.8 Example 27.8 Deduce the 1° structure of this pentapeptide

40. 27-40 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.

41. 27-41 Polypeptide Synthesis Strategy Protect the  -amino group of aa-1. Activate the  -carboxyl group of aa-1. Protect the  -carboxyl group of aa-2.

42. 27-42 Amino-Protecting Groups The most common strategy for protecting amino groups and reducing their nucleophilicity is to convert them to amides.

43. 27-43 Amino-Protecting Groups 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.

44. 27-44 Amino-Protecting Groups  The benzyloxycarbonyl group is also removed by hydrogenolysis. The intermediate carbamic acid loses carbon dioxide to give the unprotected amino group.

45. 27-45 Carboxyl-Protecting Groups  Carboxyl groups are most often protected as methyl, ethyl, or benzyl esters. Methyl and ethyl esters are prepared by Fischer esterification, and removed by hydrolysis in aqueous base under mild conditions. Benzyl esters are removed by hydrogenolysis; they are also removed by treatment with HBr in acetic acid

46. 27-46 Peptide Bond Formation  The reagent most commonly used to bring about peptide bond formation is DCC. DCC is the anhydride of a disubstituted urea and, when treated with water, is converted to DCU.

47. 27-47 Peptide Bond Formation In bringing about formation of a peptide bond, DCC acts a dehydrating.

48. 27-48 Solid-Phase Synthesis  Bruce Merrifield, 1984 Nobel Prize for Chemistry Solid support: a type of polystyrene in which about 5% of the phenyl groups carry a -CH 2 Cl group. The amino-protected C-terminal amino acid is bonded as a benzyl ester to the support beads. The polypeptide chain is then extended one amino acid at a time from the N-terminal end. When synthesis is completed, the polypeptide is released from the support beads by cleavage of the benzyl ester.

49. 27-49 Solid-Phase Synthesis  The solid support used in the Merrifield solid phase synthesis.

50. 27-50 Solid-Phase Synthesis Merrifield synthesized the enzyme ribonuclease, a protein containing 124 amino acids

51. 27-51 Peptide Bond Geometry The four atoms of a peptide bond and the two alpha carbons joined to it lie in a plane with bond angles of 120° about C and N. Model of the zwitterion form of Gly-Gly viewed from two perspectives to show the planarity of the six atoms of the peptide bond and the preferred s-trans geometry.

52. 27-52 Peptide Bond Geometry To account for this geometry, Linus Pauling proposed that a peptide bond is most accurately represented as a hybrid of two contributing structures. The hybrid has considerable C-N double bond character and rotation about the peptide bond is restricted.

53. 27-53 Peptide Bond Geometry Two conformations are possible for a planar peptide bond. Virtually all peptide bonds in naturally occurring proteins studied to date have the s-trans conformation.

54. 27-54 Secondary Structure  Secondary structure:  Secondary structure: The ordered arrangements (conformations) of amino acids in localized regions of a polypeptide or protein.  To determine from model building which conformations would be of greatest stability, Pauling and Corey assumed that: 1. All six atoms of each peptide bond lie in the same plane and in the s-trans conformation. 2. There is hydrogen bonding between the N-H group of one peptide bond and a C=O group of another peptide bond as shown in the next screen.

55. 27-55 Secondary Structure Hydrogen bonding between amide groups.

56. 27-56 Secondary Structure  On the basis of model building, Pauling and Corey proposed that two types of secondary structure should be particularly stable:  -Helix. Antiparallel  -pleated sheet.   -Helix:   -Helix: A type of secondary structure in which a section of polypeptide chain coils into a spiral, most commonly a right-handed spiral.

57. 27-57 The  -Helix The polypeptide chain is repeating units of L-alanine.

58. 27-58 The  -Helix  In a section of  -helix: There are 3.6 amino acids per turn of the helix. Each peptide bond is s-trans and planar. N-H groups of all peptide bonds point in the same direction, which is roughly parallel to the axis of the helix. C=O groups of all peptide bonds point in the opposite direction, and also parallel to the axis of the helix. The C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it. All R- groups point outward from the helix.

59. 27-59 The  -Helix  An  -helix of repeating units of L-alanine A ball-and-stick model viewed looking down the axis of the helix. A space-filling model viewed from the side.

60. 27-60  -Pleated Sheet  The antiparallel  -pleated sheet consists of adjacent polypeptide chains running in opposite directions: Each peptide bond is planar and has the s-trans conformation. The C=O and N-H groups of peptide bonds from adjacent chains point toward each other and are in the same plane so that hydrogen bonding is possible between them. All R- groups on any one chain alternate, first above, then below the plane of the sheet, etc.

61. 27-61  -Pleated Sheet  -pleated sheet with three polypeptide chains running in opposite directions (antiparallel).

62. 27-62 Tertiary Structure  Tertiary structure:  Tertiary structure: The three-dimensional arrangement in space of all atoms in a single polypeptide chain. Disulfide bonds between the side chains of cysteine play an important role in maintaining 3° structure.

63. 27-63 Tertiary Structure A ribbon model of myoglobin.

64. 27-64 Quaternary Structure  Quaternary structure:  Quaternary structure: The arrangement of polypeptide chains into a noncovalently bonded aggregation. The major factor stabilizing quaternary structure is the hydrophobic effect.  Hydrophobic effect:  Hydrophobic effect: The tendency of nonpolar groups to cluster together in such a way as to be shielded from contact with an aqueous environment.

65. 27-65 Quaternary Structure The quaternary structure of hemoglobin. The  -chains in yellow, the heme ligands in red, and the Fe atoms as white spheres.

66. 27-66 Quaternary Structure If two polypeptide chains, for example, each have one hydrophobic patch, each patch can be shielded from contact with water if the chains form a dimer.

67. 27-67 Amino Acids & Proteins End of Chapter 27