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Protein Chemistry Basics Protein function Protein structure –Primary Amino acids Linkage Protein conformation framework –Dihedral angles –Ramachandran.

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Presentation on theme: "Protein Chemistry Basics Protein function Protein structure –Primary Amino acids Linkage Protein conformation framework –Dihedral angles –Ramachandran."— Presentation transcript:

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 1.Enzymes Catalyze biological reactions 2.Structural role Cell wall Cell membrane Cytoplasm

3 Protein Structure

4

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 Xin ZhanCS 882 course project14 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)

15 Xin ZhanCS 882 course project15 Types of Turns β-turn (most common) –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

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 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 – HA

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 – HA δ-δ+δ-

19 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 Result: –Closer approach of A to H –Higher interaction energy than a simple van der Waals interaction D – HA δ-δ+δ-

20 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 NH 2 -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 (C α ) attached to: Hydrogen (H) Amino group (-NH 2 ) Carboxyl group (-COOH) Side chain (R)

29 General Amino Acid Structure CαCα H R COOHH2NH2N

30 General Amino Acid Structure At pH 7.0 CαCα H R COO-+H 3 N

31 General Amino Acid Structure

32 Amino Acids Chiral

33 Chirality: Glyceraldehyde L-glyderaldehydeD-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

45

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

52

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 –τ ABCD 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 C B D A B C D

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 –τ ABCD 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 Dihedral angle ω : rotation about the peptide bond, namely C α 1 -{C-N}- C α 2

58 Backbone Torsion Angles

59 Dihedral angle ω : rotation about the peptide bond, namely C α 1 -{C-N}- C α 2 Dihedral angle φ : rotation about the bond between N and C α

60 Backbone Torsion Angles

61 Dihedral angle ω : rotation about the peptide bond, namely C α 1 -{C-N}- C α 2 Dihedral angle φ : rotation about the bond between N and C α Dihedral angle ψ : rotation about the bond between C α and the carbonyl carbon

62 Backbone Torsion Angles

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

64

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 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 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

78

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

81

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 EnterotoxinCholera 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 CytochromeBarstar 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 “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 NH 2 -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-…..


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