1. Biochemistry Sixth Edition
ביוכימיה היא מדע החוקר את התכונות הכימיות המאפיינות מולקולות המיוצרות בתאים חיים וייחודיות להם, ומאפשרות להם לבצע את תפקידם.
Berg • Tymoczko • Stryer
Chapter 2
Protein Composition and Structure
Copyright © 2007 by W. H. Freeman and Company

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

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

5. Amino Acids
The 20 common are those used in making protein on a ribosome using mRNA and tRNA.
These are called a-amino acids since each has a carboxyl group and an amino group attached to an a-carbon atom. They differ by the sidechain or “R” group. a
+NH3-CH-COOH l R

6. 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
Acidic: Asp, Glu
7. Basic: Lys*, His*, Arg*

7. ball & stick
model
wedge
Fischer
projection

16. Amino Acid Names & Codes

17. All are Chiral at the a-carbon atom except Gly

18. 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.
+NH3 to the left = L to the right = D

19. L stereochemistry = S, for this case

20. Acids and Bases, pH, pKa
pH is defined as the negative logarithm of the concentration of H+
pH = - log [H+] = log 1/ [H+]
pKa is defined as the negative logarithm of an acid ionization constant Ka.
pKa = - log [Ka] = log 1/ [Ka]

21. Acetic Acid is a Weak Acid
Weak acids and bases do not ionize (dissociate) completely in H2O.
CH3COOH <==> CH3COO- + H+
acetic acid acetate anion
conjugate acid conjugate base form form

22. Acid Ionization Constant
[CH3COO-][H+] Ka = [CH3COOH]
[CH3COO-] -log Ka = -log [H+] + -log [CH3COOH]
[CH3COO-] pH = pKa + log [CH3COOH]

23. Henderson-Hasselbalch
Written in a more general form this expression is called the Henderson-Hassebalch equation. This relates the pH of a solution to the pKa of the weak acid in solution and the ratio of its conjugate base/conjugate acid forms.
[A-] pH = pKa + log [HA]

24. Buffers Resist Change in pH
Buffer capacity is the ability of a solution to resist changes in pH.
Most effective buffering occurs where: solution pH = buffer pKa
At this point: [conjugate acid] = [conjugate base]
Effective buffering range is usually at pH values equal to the pKa ± 1 pH unit

25. Amino Acid Ionization, pKas
Each of the 20 common a-amino acids has two pKa values, for the carboxyl group and the amino group attached to the a-carbon.
+NH3-CH2-COOH < === > +NH3-CH2-COO H+
+NH3-CH2-COO- < === > NH2-CH2-COO H+
Seven of the 20 have an ionizable sidechain and therefore have a third pKa value.

26. Amino Acid pKas a-COOH a-+NH3 R (sidechain) Gly 2.34 9.60
Ala
Val
Leu
Ile
Met
Pro
Phe
Trp
Ser
Thr
Asn
Gln
Cys
Asp
Glu
Tyr
Lys
His
Arg

27. Peptide
3.86 D, 4.25 E
Peptide
10.0

28. Effect of Change in pH

29. The Structure of an Amino Acid
An amino acid can never exist as an uncharged compound.

30. The pI of Alanine
The isoelectric point (pI) of an amino acid is the pH at which it has no net charge.

31. The pI of Lysine

32. The pI of Glutamic Acid

33. 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.

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

35. Disulfide Bond Formation

36. Disulfide bridges contribute to the overall shape of the protein.

37. Straight and Curly Hair

38. Insulin Insulin has two interchain disulfide bridges and
one intrachain disulfide bridge.

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

40. Peptide Bond Planarity
6 atoms are coplanar

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

42. Rotation Sites, y and f
The rotational arrangements about a-carbons of a peptide or protein gives its secondary (2o) structure.

43. f
View from N-term
to a-carbon

44. y
View from a-carbon to C-term

45. Ramachandran Plot

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

47. a-helix in a Ramachandran Plot
y = -47
f = -57

48. The a-helix, a helix

49. The a-helix

50. Hydrogen Bond Contacts

51. a-helix in a Protein

52. b-sheet in a Ramachandran Plot
y = +135
f = -139

53. A b-sheet strand

54. Anti-parallel b-sheet

55. Parallel b-sheet

56. Anti-parallel b-sheet

57. b-sheet in a Protein

58. b-turn or hairpin turn A turn is 4-5 aa residues.
A b-turn (hairpin) is
4 aa residues.

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

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

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

62. Tertiary Structure (3o)
The three dimensional folding of a polypeptide is its tertiary structure.
Both the a-helix and b-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 = charged Yellow = hydrophobic

65. Porin, a membrane spanning protein

66. Domain A domain is a discrete globular area within protein.
There are four general types:
All a only a- helices and loops
All b only b- sheet & loops or turns
Mixed a/b alternating abab
a+b cluster of a then cluster of b

67. Domain
Multiple domains exist in the protein below.

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

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

70. Denaturing Proteins Denaturing agents destroy the protein 3o 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.

71. Denaturing agents

72. Disulfide oxidation-reduction
Reduced
Oxidized

73. Ribonuclease
4 disulfide
bonds.
-S-S-

74. Then removing urea and
ME permits reoxidation.

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

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

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

78. Protein Cleavage Protein sequencing is most manageable with
small polypeptides.
Therefore, in order to sequence a large protein,
it must be cleaved into smaller pieces.
Cleavage is conducted using either chemical or
enzymatic methods.
The pieces must be separated and purified
before sequencing.

79. Chemical and Enzymatic Cleavage

80. CNBr Cleavage at Met

81. Enzymatic cleavage by Trypsin

82. הערות: טריפסין חותך בצד הקרבוקסילי של : Lys, Arg
שאלה: לפניך תוצרי הביקוע של פפטיד באמצעות טריפסין וכימוטריפסין:
טריפסין:
Phe-Val-Ser-Arg
Met-Asn-Lys
Gly-Trp
כימוטריפסין:
Val-Ser-Arg-Gly-Trp
Met-Asn-Lys-Phe
הערות: טריפסין חותך בצד הקרבוקסילי של : Lys, Arg
וכימוטריפסין חותך בצד הקרבוקסילי של : Phe,Trp,Tyr.
מהו רצף החומצות האמיניות בפפטיד.
תשובה:
Met-Asn-Lys-Phe-Val-Ser-Arg-Gly-Trp

83. An Example, Peptide overlap