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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 Protein Function in Cell 1. Enzymes • Catalyze biological reactions 2. Structural role • • • Cell wall Cell membrane Cytoplasm Protein Structure Protein Structure Model Molecule: Hemoglobin Hemoglobin: Background • Protein in red blood cells Red Blood Cell (Erythrocyte) 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 Heme Groups in Hemoglobin 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) Hemoglobin – Quaternary Structure Two alpha subunits and two beta subunits (141 AA per alpha, 146 AA per beta) Hemoglobin – Tertiary Structure One beta subunit (8 alpha helices) Hemoglobin – Secondary Structure alpha helix 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 14 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 Xin Zhan CS 882 course project 15 Structure Stabilizing Interactions • Noncovalent – Van der Waals forces (transient, weak electrical attraction of one atom for another) – Hydrophobic (clustering of nonpolar groups) – Hydrogen bonding 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–H A 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 δ- δ+ δ- D–H A 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 δ- • Result: δ+ δ- D–H A – Closer approach of A to H – Higher interaction energy than a simple van der Waals interaction Hydrogen Bonding And Secondary Structure alpha-helix beta-sheet 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 Disulfide Bonds • Side chain of cysteine contains highly reactive thiol group • Two thiol groups form a disulfide bond Disulfide Bridge 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 Disulfide Bridge – Linking Distant Amino Acids Hemoglobin – Primary Structure NH2-Val-His-Leu-Thr-Pro-Glu-Glu- Lys-Ser-Ala-Val-Thr-Ala-Leu-TrpGly-Lys-Val-Asn-Val-Asp-Glu-ValGly-Gly-Glu-….. beta subunit amino acid sequence Protein Structure - Primary • Protein: chain of amino acids joined by peptide bonds 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) General Amino Acid Structure H H2N α C R COOH General Amino Acid Structure At pH 7.0 H +H3N α C R COO- General Amino Acid Structure Amino Acids • Chiral Chirality: Glyceraldehyde D-glyderaldehyde L-glyderaldehyde Amino Acids • Chiral • 20 naturally occuring; distinguishing side chain 20 Naturally-occurring Amino Acids Amino Acids • Chiral • 20 naturally occuring; distinguishing side chain • Classification: • Non-polar (hydrophobic) • Charged polar • Uncharged polar Alanine: Nonpolar Serine: Uncharged Polar Aspartic Acid Charged Polar Glycine Nonpolar (special case) Peptide Bond • Joins amino acids Peptide Bond Formation Peptide Chain Peptide Bond • Joins amino acids • 40% double bond character – Caused by resonance Peptide bond • Joins amino acids • 40% double bond character – Caused by resonance – Results in shorter bond length Peptide Bond Lengths Peptide bond • Joins amino acids • 40% double bond character – Caused by resonance – Results in shorter bond length – Double bond disallows rotation Protein Conformation Framework • Bond rotation determines protein folding, 3D structure Bond Rotation Determines Protein Folding 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 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 Ethane Rotation A D B C A D B C 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 Backbone Torsion Angles Backbone Torsion Angles • Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2 Backbone Torsion Angles 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 Backbone Torsion Angles 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 Backbone Torsion Angles 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 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 Backbone Torsion Angles 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 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 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 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 Ramachandran Plot • Plot of φ vs. ψ • The computed angles which are sterically allowed fall on certain regions of plot Computed Ramachandran Plot White = sterically disallowed conformations (atoms come closer than sum of van der Waals radii) Blue = sterically allowed conformations Ramachandran Plot • Plot of φ vs. ψ • Computed sterically allowed angles fall on certain regions of plot • Experimentally determined angles fall on same regions Experimental Ramachandran Plot φ, ψ distribution in 42 high-resolution protein structures (x-ray crystallography) 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 alphahelix) 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 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 Ramachandran Plot And Secondary Structure • White = sterically disallowed conformations • Red = sterically allowed regions if strict (greater) radii are used (namely righthanded alpha helix and beta sheet) • Yellow = sterically allowed if shorter radii are used (i.e. atoms allowed closer together; brings out left-handed helix) Sample Ramachandran Plot Alanine Ramachandran Plot Arginine Ramachandran Plot Glutamine Ramachandran Plot Glycine Ramachandran Plot Note more allowed regions due to less steric hindrance - Turns Proline Ramachandran Plot Note less allowed regions due to structure rigidity φ, ψ and Secondary Structure Name φ ψ Structure ------------------- ------- ------- --------------------------------alpha-L 57 47 left-handed alpha helix 3-10 Helix -49 -26 right-handed. π helix -57 -80 right-handed. Type II helices -79 150 left-handed helices formed by polyglycine and polyproline. Collagen -51 153 right-handed coil formed of three left handed helicies. Sequence Similarity • Sequence similarity implies structural, functional, and evolutionary commonality Homologous Proteins: Enterotoxin and Cholera toxin Enterotoxin Cholera toxin 80% homology Sequence Similarity • Sequence similarity implies structural, functional, and evolutionary commonality • Low sequence similarity implies little structural similarity Nonhomologous Proteins: Cytochrome and Barstar Cytochrome Barstar Less than 20% homology 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! Sequence Similarity Exception • Sickle-cell anemia resulting from one residue change in hemoglobin protein • Replace highly polar (hydrophilic) glutamate with nonpolar (hydrophobic) valine Sickle-cell mutation in hemoglobin sequence 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 Sickle-cell Trait • Oxy-hemoglobin is isolated, but deoxyhemoglobin sticks together in polymers, distorting RBC • Some cells take on “sickle” shape Sickle-cell 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 Hemoglobin Polymerization Normal Mutant 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 Capillary Blockage Sickle-cell Trait • Oxy-hemoglobin is isolated, but deoxyhemoglobin 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 Protein: The Machinery of Life NH2-Val-His-Leu-Thr-Pro-Glu-GluLys-Ser-Ala-Val-Thr-Ala-Leu-TrpGly-Lys-Val-Asn-Val-Asp-Glu-ValGly-Gly-Glu-….. “Life is the mode of existence of proteins, and this mode of existence essentially consists in the constant selfrenewal of the chemical constituents of these substances.” Friedrich Engles, 1878