1. Protein Chemistry Basics
Protein function
Protein structure
Primary
Amino acids
Linkage
Protein conformation framework
Dihedral angles
Ramachandran plots
Sequence similarity and variation

2. Protein Function in Cell
Enzymes
Catalyze biological reactions
Structural role
Cell wall
Cell membrane
Cytoplasm

3. Protein Structure

4. Protein Structure

5. Model Molecule: Hemoglobin

6. Hemoglobin: Background
Protein in red blood cells

7. Red Blood Cell (Erythrocyte)

8. Hemoglobin: Background
Protein in red blood cells
Composed of four subunits, each containing a heme group: a ring-like structure with a central iron atom that binds oxygen

9. Heme Groups in Hemoglobin

10. Hemoglobin: Background
Protein in red blood cells
Composed of four subunits, each containing a heme group: a ring-like structure with a central iron atom that binds oxygen
Picks up oxygen in lungs, releases it in peripheral tissues (e.g. muscles)

11. Hemoglobin – Quaternary Structure
Two alpha subunits and two beta subunits
(141 AA per alpha, 146 AA per beta)

12. Hemoglobin – Tertiary Structure
One beta subunit (8 alpha helices)

13. Hemoglobin – Secondary Structure
alpha helix

14. β-Hairpin Motif
Simplest protein motif involving two beta strands [from Wikipedia]
adjacent in primary sequence
antiparallel
linked by a short loop
As isolated ribbon or part of beta sheet
a special case of a turn
direction of protein backbone reverses
flanking secondary structure elements interact (hydrogen bonds)
Xin Zhan
CS 882 course project

15. Types of Turns β-turn (most common) δ-turn γ-turn α-turn π-turn ω-loop
donor and acceptor residues of hydrogen bonds are separated by 3 residues (i i +3 H-bonding)
δ-turn
i i +1 H-bonding
γ-turn
i i +2 H-bonding
α-turn
i i +4 H-bonding
π-turn
i i +5 H-bonding
ω-loop
a longer loop with no internal hydrogen bonding
Is characterized by…
1 Delta ; 2 gamma
People have found beta and pi turn occurring in beta hairpin
Xin Zhan
CS 882 course project

16. Structure Stabilizing Interactions
Noncovalent
Van der Waals forces (transient, weak electrical attraction of one atom for another)
Hydrophobic (clustering of nonpolar groups)
Hydrogen bonding

17. Hydrogen Bonding D – H A Involves three atoms:
Donor electronegative atom (D)
(Nitrogen or Oxygen in proteins)
Hydrogen bound to donor (H)
Acceptor electronegative atom (A) in close proximity
D – H A

18. D-H Interaction
Polarization due to electron withdrawal from the hydrogen to D giving D partial negative charge and the H a partial positive charge
Proximity of the Acceptor A causes further charge separation
D – H A δ- δ+

19. D-H Interaction D – H A δ- δ+
Polarization due to electron withdrawal from the hydrogen to D giving D partial negative charge and the H a partial positive charge
Proximity of the Acceptor A causes further charge separation
Result:
Closer approach of A to H
Higher interaction energy than a simple van der Waals interaction
D – H A δ- δ+

20. And Secondary Structure
Hydrogen Bonding
And Secondary Structure
alpha-helix
beta-sheet

21. Structure Stabilizing Interactions
Noncovalent
Van der Waals forces (transient, weak electrical attraction of one atom for another)
Hydrophobic (clustering of nonpolar groups)
Hydrogen bonding
Covalent
Disulfide bonds

22. Disulfide Bonds
Side chain of cysteine contains highly reactive thiol group
Two thiol groups form a disulfide bond

23. Disulfide Bridge

24. Disulfide Bonds
Side chain of cysteine contains highly reactive thiol group
Two thiol groups form a disulfide bond
Contribute to the stability of the folded state by linking distant parts of the polypeptide chain 

25. Disulfide Bridge – Linking Distant Amino Acids

26. Hemoglobin – Primary Structure
NH2-Val-His-Leu-Thr-Pro-Glu-Glu-
Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp-
Gly-Lys-Val-Asn-Val-Asp-Glu-Val-
Gly-Gly-Glu-…..
beta subunit amino acid sequence

27. Protein Structure - Primary
Protein: chain of amino acids joined by peptide bonds

28. Protein Structure - Primary
Protein: chain of amino acids joined by peptide bonds
Amino Acid
Central carbon (Cα) attached to:
Hydrogen (H)
Amino group (-NH2)
Carboxyl group (-COOH)
Side chain (R)

29. General Amino Acid StructureH
H2N Cα
COOH R

30. General Amino Acid Structure
At pH 7.0 H
+H3N Cα
COO- R

31. General Amino Acid Structure

32. Amino Acids
Chiral

33. Chirality: Glyceraldehyde
D-glyderaldehyde
L-glyderaldehyde

34. Amino Acids
Chiral
20 naturally occuring; distinguishing side chain

35. 20 Naturally-occurring Amino Acids

36. Amino Acids Chiral 20 naturally occuring; distinguishing side chain
Classification:
Non-polar (hydrophobic)
Charged polar
Uncharged polar

37. Alanine:
Nonpolar

38. Serine:
Uncharged Polar

39. Aspartic Acid Charged Polar

40. Glycine Nonpolar (special case)

41. Peptide Bond
Joins amino acids

42. Peptide Bond Formation

43. Peptide Chain

44. Peptide Bond Joins amino acids 40% double bond character
Caused by resonance

46. Peptide bond Joins amino acids 40% double bond character
Caused by resonance
Results in shorter bond length

47. Peptide Bond Lengths

48. Peptide bond Joins amino acids 40% double bond character
Caused by resonance
Results in shorter bond length
Double bond disallows rotation

49. Protein Conformation Framework
Bond rotation determines protein folding, 3D structure

50. Bond Rotation Determines Protein Folding

51. Protein Conformation Framework
Bond rotation determines protein folding, 3D structure
Torsion angle (dihedral angle) τ
Measures orientation of four linked atoms in a molecule: A, B, C, D

53. Protein Conformation Framework
Bond rotation determines protein folding, 3D structure
Torsion angle (dihedral angle) τ
Measures orientation of four linked atoms in a molecule: A, B, C, D
τABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D

54. Ethane Rotation A A D D B B C C

55. Protein Conformation Framework
Bond rotation determines protein folding, 3D structure
Torsion angle (dihedral angle) τ
Measures orientation of four linked atoms in a molecule: A, B, C, D
τABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D
Three repeating torsion angles along protein backbone: ω, φ, ψ

56. Backbone Torsion Angles

57. Backbone Torsion Angles
Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2

58. Backbone Torsion Angles

59. Backbone Torsion Angles
Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2
Dihedral angle φ : rotation about the bond between N and Cα

60. Backbone Torsion Angles

61. Backbone Torsion Angles
Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2
Dihedral angle φ : rotation about the bond between N and Cα
Dihedral angle ψ : rotation about the bond between Cα and the carbonyl carbon

62. Backbone Torsion Angles

63. Backbone Torsion Angles
ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl π electrons and nitrogen lone pair

65. Backbone Torsion Angles
ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair
φ and ψ are flexible, therefore rotation occurs here

66. Backbone Torsion Angles

67. Backbone Torsion Angles
ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair
φ and ψ are flexible, therefore rotation occurs here
However, φ and ψ of a given amino acid residue are limited due to steric hindrance

68. Steric Hindrance
Interference to rotation caused by spatial arrangement of atoms within molecule
Atoms cannot overlap
Atom size defined by van der Waals radii
Electron clouds repel each other

69. Backbone Torsion Angles
ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair
φ and ψ are flexible, therefore rotation occurs here
However, φ and ψ of a given amino acid residue are limited due to steric hindrance
Only 10% of the {φ, ψ} combinations are generally observed for proteins
First noticed by G.N. Ramachandran

70. G.N. Ramachandran
Used computer models of small polypeptides to systematically vary φ and ψ with the objective of finding stable conformations
For each conformation, the structure was examined for close contacts between atoms
Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii
Therefore, φ and ψ angles which cause spheres to collide correspond to sterically disallowed conformations of the polypeptide backbone

71. Ramachandran Plot Plot of φ vs. ψ
The computed angles which are sterically allowed fall on certain regions of plot

72. Computed Ramachandran Plot
White = sterically disallowed conformations (atoms come closer than sum of van der Waals radii)
Blue = sterically allowed conformations

73. Ramachandran Plot Plot of φ vs. ψ
Computed sterically allowed angles fall on certain regions of plot
Experimentally determined angles fall on same regions

74. Experimental Ramachandran Plot
φ, ψ distribution in 42 high-resolution protein structures (x-ray crystallography)

75. And Secondary Structure
Ramachandran Plot
And Secondary Structure
Repeating values of φ and ψ along the chain result in regular structure
For example, repeating values of φ ~ -57° and ψ ~ -47° give a right-handed helical fold (the alpha-helix)

76. The structure of cytochrome C shows many segments of helix and the Ramachandran plot shows a tight grouping of φ, ψ angles near -50,-50
alpha-helix
cytochrome C
Ramachandran plot

77. Similarly, repetitive values in the region of φ = -110 to –140 and ψ = +110 to +135 give beta sheets. The structure of plastocyanin is composed mostly of beta sheets; the Ramachandran plot shows values in the
–110, +130 region:
beta-sheet
plastocyanin
Ramachandran plot

79. Ramachandran Plot And Secondary Structure
White = sterically disallowed conformations
Red = sterically allowed regions if strict (greater) radii are used (namely right-handed alpha helix and beta sheet)
Yellow = sterically allowed if shorter radii are used (i.e. atoms allowed closer together; brings out left-handed helix)

80. Sample Ramachandran Plot

82. Alanine Ramachandran Plot

83. Arginine Ramachandran Plot

84. Glutamine Ramachandran Plot

85. Glycine Ramachandran Plot
Note more allowed regions due to less steric hindrance - Turns

86. Proline Ramachandran Plot
Note less allowed regions due to structure rigidity

87. φ, ψ and Secondary Structure
Name φ ψ Structure
alpha-L left-handed alpha helix
3-10 Helix right-handed.
π helix right-handed.
Type II helices left-handed helices
formed by polyglycine
and polyproline.
Collagen right-handed coil formed
of three left handed
helicies.

88. Sequence Similarity
Sequence similarity implies structural, functional, and evolutionary commonality

89. Homologous Proteins: Enterotoxin and Cholera toxin
80% homology

90. Sequence Similarity
Sequence similarity implies structural, functional, and evolutionary commonality
Low sequence similarity implies little structural similarity

91. Nonhomologous Proteins: Cytochrome and Barstar
Less than 20% homology

92. Sequence Similarity
Sequence similarity implies structural, functional, and evolutionary commonality
Low sequence similarity implies little structural similarity
Small mutations generally well-tolerated by native structure – with exceptions!

93. Sequence Similarity Exception
Sickle-cell anemia resulting from one residue change in hemoglobin protein
Replace highly polar (hydrophilic) glutamate with nonpolar (hydrophobic) valine

94. Sickle-cell mutation in hemoglobin sequence

95. Normal Trait
Hemoglobin molecules exist as single, isolated units in RBC, whether oxygen bound or not
Cells maintain basic disc shape, whether transporting oxygen or not

96. Sickle-cell Trait
Oxy-hemoglobin is isolated, but de-oxyhemoglobin sticks together in polymers, distorting RBC
Some cells take on “sickle” shape

97. Sickle-cell

98. RBC Distortion Hydrophobic valine replaces hydrophilic glutamate
Causes hemoglobin molecules to repel water and be attracted to one another
Leads to the formation of long hemoglobin filaments

99. Hemoglobin Polymerization
Normal
Mutant

100. RBC Distortion Hydrophobic valine replaces hydrophilic glutamate
Causes hemoglobin molecules to repel water and be attracted to one another
Leads to the formation of long hemoglobin filaments
Filaments distort the shape of red blood cells (analogy: icicle in a water balloon)
Rigid structure of sickle cells blocks capillaries and prevents red blood cells from delivering oxygen

101. Capillary Blockage

102. Sickle-cell Trait
Oxy-hemoglobin is isolated, but de-oxyhemoglobin sticks together in polymers, distorting RBC
Some cells take on “sickle” shape
When hemoglobin again binds oxygen, again becomes isolated
Cyclic alteration damages hemoglobin and ultimately RBC itself

103. Protein: The Machinery of Life
NH2-Val-His-Leu-Thr-Pro-Glu-Glu-
Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp-
Gly-Lys-Val-Asn-Val-Asp-Glu-Val-
Gly-Gly-Glu-…..
“Life is the mode of existence of proteins, and this mode of existence essentially consists in the constant self-renewal of the chemical constituents of these substances.”
Friedrich Engles, 1878