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Protein Chemistry Basics
Protein function Protein structure Primary Amino acids Linkage Protein conformation framework Dihedral angles Ramachandran plots Sequence similarity and variation
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Protein Function in Cell
Enzymes Catalyze biological reactions Structural role Cell wall Cell membrane Cytoplasm
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Protein Structure
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Protein Structure
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Model Molecule: Hemoglobin
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Hemoglobin: Background
Protein in red blood cells
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Red Blood Cell (Erythrocyte)
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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
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Heme Groups in Hemoglobin
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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)
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Hemoglobin – Quaternary Structure
Two alpha subunits and two beta subunits (141 AA per alpha, 146 AA per beta)
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Hemoglobin – Tertiary Structure
One beta subunit (8 alpha helices)
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Hemoglobin – Secondary Structure
alpha helix
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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
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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
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Structure Stabilizing Interactions
Noncovalent Van der Waals forces (transient, weak electrical attraction of one atom for another) Hydrophobic (clustering of nonpolar groups) Hydrogen bonding
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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
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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+
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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+
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And Secondary Structure
Hydrogen Bonding And Secondary Structure alpha-helix beta-sheet
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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
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Disulfide Bonds Side chain of cysteine contains highly reactive thiol group Two thiol groups form a disulfide bond
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Disulfide Bridge
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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
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Disulfide Bridge – Linking Distant Amino Acids
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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
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Protein Structure - Primary
Protein: chain of amino acids joined by peptide bonds
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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)
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General Amino Acid Structure
H H2N Cα COOH R
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General Amino Acid Structure
At pH 7.0 H +H3N Cα COO- R
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General Amino Acid Structure
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Amino Acids Chiral
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Chirality: Glyceraldehyde
D-glyderaldehyde L-glyderaldehyde
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Amino Acids Chiral 20 naturally occuring; distinguishing side chain
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20 Naturally-occurring Amino Acids
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Amino Acids Chiral 20 naturally occuring; distinguishing side chain
Classification: Non-polar (hydrophobic) Charged polar Uncharged polar
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Alanine: Nonpolar
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Serine: Uncharged Polar
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Aspartic Acid Charged Polar
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Glycine Nonpolar (special case)
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Peptide Bond Joins amino acids
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Peptide Bond Formation
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Peptide Chain
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Peptide Bond Joins amino acids 40% double bond character
Caused by resonance
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Peptide bond Joins amino acids 40% double bond character
Caused by resonance Results in shorter bond length
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Peptide Bond Lengths
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Peptide bond Joins amino acids 40% double bond character
Caused by resonance Results in shorter bond length Double bond disallows rotation
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Protein Conformation Framework
Bond rotation determines protein folding, 3D structure
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Bond Rotation Determines Protein Folding
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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
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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
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Ethane Rotation A A D D B B C C
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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
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Backbone Torsion Angles
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Backbone Torsion Angles
Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2
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Backbone Torsion Angles
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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
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Backbone Torsion Angles
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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
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Backbone Torsion Angles
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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
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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
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Backbone Torsion Angles
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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
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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
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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
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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
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Ramachandran Plot Plot of φ vs. ψ
The computed angles which are sterically allowed fall on certain regions of plot
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Computed Ramachandran Plot
White = sterically disallowed conformations (atoms come closer than sum of van der Waals radii) Blue = sterically allowed conformations
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Ramachandran Plot Plot of φ vs. ψ
Computed sterically allowed angles fall on certain regions of plot Experimentally determined angles fall on same regions
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Experimental Ramachandran Plot
φ, ψ distribution in 42 high-resolution protein structures (x-ray crystallography)
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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)
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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
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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
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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)
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Sample Ramachandran Plot
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Alanine Ramachandran Plot
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Arginine Ramachandran Plot
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Glutamine Ramachandran Plot
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Glycine Ramachandran Plot
Note more allowed regions due to less steric hindrance - Turns
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Proline Ramachandran Plot
Note less allowed regions due to structure rigidity
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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.
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Sequence Similarity Sequence similarity implies structural, functional, and evolutionary commonality
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Homologous Proteins: Enterotoxin and Cholera toxin
80% homology
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Sequence Similarity Sequence similarity implies structural, functional, and evolutionary commonality Low sequence similarity implies little structural similarity
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Nonhomologous Proteins: Cytochrome and Barstar
Less than 20% homology
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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!
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Sequence Similarity Exception
Sickle-cell anemia resulting from one residue change in hemoglobin protein Replace highly polar (hydrophilic) glutamate with nonpolar (hydrophobic) valine
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Sickle-cell mutation in hemoglobin sequence
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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
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Sickle-cell Trait Oxy-hemoglobin is isolated, but de-oxyhemoglobin sticks together in polymers, distorting RBC Some cells take on “sickle” shape
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Sickle-cell
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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
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Hemoglobin Polymerization
Normal Mutant
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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
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Capillary Blockage
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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
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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
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