1. Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition Chapter 2 Protein Composition and Structure

2. Functions of some Proteins Catalysis (enzymes) Structural (collagen) Contractile (muscle) Transport (hemoglobin) Storage (myoglobin) Electron transport (cytochromes) Hormones (insulin) Growth factor (EGF) DNA binding (histones) Ribosomal proteins Toxins and venoms (cholera & melittin) Vision (opsins) Immunoglobins

3. Replication protein around DNA

4. ConformationaI change Lactoferrin

5. Levels of Protein Structure Primary structure (1 o ) – sequence of amino acids starting from the N-terminus of the peptide. Secondary structure (2 o ) – conformations of the peptide chain from rotation about the  -Cs, e.g.  -helices and  -sheets, etc. Tertiary structure (3 o ) – three dimensional shape of the fully folded polypeptide chain. Quaternary structure (4 o ) - arrangement of two or more protein chains into multisubunit molecule

6. Levels of Protein Structure Bonding: 1 o = covalent 2 o = H-bond 3 o = covalent & noncovalent 4 o = noncovalent

7. Amino Acids The 20 common are those used in making protein on a ribosome using mRNA and tRNA. These are called  -amino acids since each has a carboxyl group and an amino group attached to an  -carbon atom. They differ by the sidechain or “R” group.  + NH 3 -CH-COOH l R

8. Amino Acid Classification Classification is made using the structure of the side chain, R. (* = essential) 1. None (hydrogen):Gly 2. Non-polar: Aliphatic:Ala, Val*, Leu*, Ile*, Met* Alicyclic:Pro 3. Aromatic:Phe*, Tyr, Trp* 4. Polar uncharged:Ser, Thr*, Asn, Gln 5. Thiol:Cys 6.Acidic:Asp, Glu 7. Basic:Lys*, His*, Arg*

9. ball & stick model wedge Fischer projection

18. 1 o aliphatic pKa = 13.6 Aromatic pKa = 7.05 Cl - chloride

20. Amino Acid Names & Codes

21. Other structures

22. All are Chiral at the  -carbon atom except Gly

23. D, L Assignments The convention for making D, L assignments is to draw a Fischer projection with the carboxyl at the top and the R at the bottom. + NH 3 to the left = L to the right = D

24. L stereochemistry = S, for this case

25. Amino Acid Ionization, pKas Each of the 20 common  -amino acids has two pKa values, for the carboxyl group and the amino group attached to the  -carbon. + NH 3 -CH 2 -COOH + NH 3 -CH 2 -COO - + H + + NH 3 -CH 2 -COO - NH 2 -CH 2 -COO - + H + Seven of the 20 have an ionizable sidechain and therefore have a third pKa value.

26. Amino Acid pKas  -COOH  - + NH 3 R (sidechain) Gly2.349.60 Ala2.349.69 Val2.329.62 Leu2.369.60 Ile 2.369.60 Met2.289.21 Pro 1.9910.60 Phe1.839.13 Trp 2.839.39 Ser 2.219.15 Thr 2.6310.43 Asn2.028.80 Gln 2.179.13 Cys 1.7110.788.33 Asp 2.099.823.86 Glu 2.199.674.25 Tyr 2.209.1110.07 Lys 2.188.9510.79 His 1.829.176.00 Arg2.179.0412.48

27. 3.86 D, 4.25 E 10.0 Peptide

28. Effect of Change in pH

29. Formation of a Peptide Two amino acids are joined together to form a peptide (amide) bond with a loss of HOH. After becoming part of a peptide or protein these are called “residues” due to loss of HOH.

30. Primary Structure (1 o ) The sequence of amino acids (N-term to C-term) in a peptide or protein is its primary structure. A pentapeptide

31. Convention for writing a peptide Dashes indicate a knowledge of the sequence of amino acid residues. N-term Tyr-Gly-Asp-Phe-Leu C-term Commas indicate that the residue is known but not the sequence. N-term Tyr,Gly,Asp,Phe,Leu C-term Asx indicates that the residue could be either Asp or Asn (The same applies to Glx). N-term Tyr-Gly-Asx-Phe-Leu C-term

32. Disulfide Bond Formation

33. Disulfide Bonds in Insulin

34. Peptide Bond Resonance.. Due to resonance participation of the unshared pair of electrons on N, amides are neutral.

35. Peptide Bond Planarity 6 atoms are coplanar

36. Peptide Bond Structures Less crowded in the favored trans arrangement

37. A prolyl residue Crowded in both cis and trans arrangements

38. Rotation Sites,  and  The rotational arrangements about  -carbons of a peptide or protein gives its secondary (2 o ) structure.

39.  View from N-term to  -carbon

40.  View from  -carbon to C-term

41. Ramachandran Plot

42. Secondary Structure (2 o ) The two most common secondary structures are the  -helix and the  -sheet. Each of these 2 o structures have fairly specific  and  angles. All other rotational angles represent “random” secondary structure. Secondary structure is maintained by hydrogen bonding.  -helix by intramolecular H-bonds  -sheet by intermolecular H-bonds

43.  -helix in a Ramachandran Plot  = -47  = -57

44. The  -helix, a 3.6 13 helix

45. The  -helix

46. Hydrogen Bond Contacts

47.  -helix in a Protein

48.  -sheet in a Ramachandran Plot  = +135  = -139

49. A  -sheet strand

50. Anti-parallel  -sheet

51. Parallel  -sheet

52. Anti-parallel  -sheet

53.  -sheet structures

54.  -sheet in a Protein

55.       

56.  -turn or hairpin turn A turn is 4-5 aa residues. A  -turn (hairpin) is 4 aa residues.

57. Loops are usually larger than turns. >5 aa residues

58.  -Keratin, a fibrous protein, forms an  coiled coil Each strand is a modified  -helix, 3.5 residues/turn. Right-handed helices form a left-handed supercoil.

59. A heptad repeat

60. Collagen, a triple helix Gly every third residue; Gly-Pro-HPro is frequent. Interstrand H-bond at Gly. No Cys, so, no -S-S-

61. Collagen sequences

62. Tertiary Structure (3 o ) The three dimensional folding of a polypeptide is its tertiary structure. Both the  -helix and  -sheet may exist within the tertiary structure. Generally the distribution of amino acid sidechains in a globular protein finds mostly nonpolar residues in the interior of the protein and polar residues on the surface. Tertiary structure is maintained by noncovalent interactions and disulfide bonding.

63. Myoglobin, a globular protein

64. Myoglobin Surface Cross-section Blue = chargedYellow = hydrophobic

65. Porin, a membrane spanning protein

66. Motifs, (Supersecondary structure) These are combinations of secondary structures observed in different proteins. Examples: Helix-loop-helix Coiled coil Helix bundle   - hairpin  - meander Greek key  - sandwich

67. Motifs

68. Domain A domain is a discrete globular area within protein. There are four general types: All  only  -  helices and loops All   only  - sheet & loops or turns Mixed  alternating   cluster of  then cluster of 

69. Domain Multiple domains exist in the protein below.

70. Quaternary Structure (4 o ) is an assembly of 3 o structures (two or more subunits). A dimer

71. Hemoglobin, a tetramer Quaternary structure is maintained by noncovalent interactions

72. Denaturing Proteins Denaturing agents destroy the protein 3 o structure (causes the protein to unravel). Methods: Heat; Extremes of pH; Detergents; Mechanical agitation; Mercaptoethanol – breaks -S-S- bonds; 6M guanidine HCl or 10M urea: these are chaotropic agents that break up noncovalent interactions.

73. Denaturing agents

74. Disulfide oxidation-reduction Oxidized Reduced

75. Ribonuclease 4 disulfide bonds. -S-S-

76. Then removing urea and ME permits reoxidation.

77. A trace of ME allows reduction/oxidation to occur until the low free energy form is found and >98% of activity is restored. Reoxidation reforms –S-S- but not necessarily in the correct place.

78. Disulfide Bonds in Insulin Reduction and treatment as with ribonuclease gives <1% restored activity.

79. Sharp Transition suggests an all or nothing effect in denaturing.

80. Protein Folding Protein folding typically occurs as the protein comes off of the ribosome. It is cooperative process and is driven in part by the hydrophobic effect to reach a low free energy conformation. Folding is assisted by molecular chaperones and protein disufide isomerase.

81. Protein Folding The cooperative process is such that the initial formation of small elements of structure accelerate subsequent structure development (folding). In folding, the polypeptide chain goes from a high energy, high entropy state to a low energy, low entropy state. This occurs faster in vivo that in vitro. Molecular chaperones serve to prevent most errors in folding and when such occurs the assist in refolding. Protein disufide isomerase preventing errors in –S-S- Formation and in correcting errors that occur.

82. Modified amino acid residues Modification occurs after a protein is synthesized.

83. Modified amino acid residues Modification produces fluorescence in jellyfish.

84. Lysozyme, an enzyme Space filling Ball & stick

85. Lysozyme Protein backbone

86. Lysozyme Ribbon diagrams 3 o structure Active site & -S-S-

87. End of Chapter 2 Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition