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Biochemistry 330 September 13, 2011 BioChem 330 - Course Outline • Bio-molecular Structure/Function (I) – PROTEINS • Structure – – – – Chemistry of amino acid building blocks Primary, secondary and tertiary structure Protein folding, thermodynamics and kinetics Predictions of protein folding, dynamics • Function – Binding ….a tale of two globins (hemoglobin and immunoglobulin) • Chemistry of amino acids building blocks – Chirality – Structure – Functionality – Acid/base behavior – Polarity • The Peptide Bond How do we measure hydrophobicity? • Polarity Scale ranges from +1.18 (K) to -1.68 (I) – suggests which amino acids R groups are likely to be exposed to solvent (pos value) and which are likely to be buried in interior of protein (neg value) – Based on calculations of the energy necessary to transfer the side group from water to a less polar solvent. – Guy, Biophysical J. 1984, 47, 61-70 • Hydropathy Scale ranges from -4.5(R) to +4.5 (I) – Essentially the opposite of polarity scale, pos numbers likely buried and neg numbers likely to be exposed. – Based on analysis of where side groups tend to be found – Kyte and Doolittle, J. Mol. Bio, 1982 157, 105-132 Thermodynamic properties of amino acids Name Polarity Hydropathy pKa-COOH pKa-NH2 pKa-R Aspartate Asp - D +0.80 -3.5 2.09 9.82 3.86 Glutamate Glu- E +0.77 -3.5 2.19 9.67 4.25 Histidine His – H +1.18 -3.9 2.18 8.95 10.5 Lysine Lys – K -0.49 -3.2 1.82 9.17 6.00 Arginine Arg - R +0.84 -4.5 2.17 9.04 12.5 Thermodynamic properties (cont’d) Name Polarity Hydropathy pKa-COOH pKa-NH2 Alanine Ala - A +0.06 +1.8 2.34 9.69 Valine Val- V -1.09 +4.2 2.32 9.62 Leucine Leu – L -1.21 +3.8 2.36 9.60 Isoleucine Ile – I -1.31 +4.5 2.36 9.68 Proline Pro - P +0.70 - 1.6 1.99 10.96 Phenylalanine Phe - F -1.68 +2.8 1.83 9.13 Tryptophan Trp - W -0.88 -0.90 2.38 9.39 Methionine Met - M -1.23 +1.9 2.38 9.21 pKa-R Thermodynamic properties (cont’d) Name Polarity Hydropathy pKa-COOH pKa-NH2 pKa-R Glyine Gly - G +0.41 -0.40 2.34 9.60 Serine Ser- S +0.50 -0.80 2.21 9.15 13.6 Threonine Thr - T +.27 -0.70 2.63 10.4 13.6 Cysteine Cys - C -1.36 +2.5 1.71 10.8 8.3 Tyrosine Tyr - Y -0.33 - 1.3 2.20 9.11 10.5 Asparagine Asn - N +0.48 -3.5 2.02 8.80 Glutamine Gln - Q +0.73 -3.5 2.17 9.13 Amino acids can be post-translationally modified • Amino or carboxyl group can be modified • sugars, fats, methyl, phosphate, sulfate groups can be added to side chain • Modifications change the way the amino acid behaves The polymer Amino Acids are joined together using Peptide Bonds to make Proteins • Peptide bonds link amino acids together through -NH2 on one amino acid and the – COOH on another. • in vivo translation is process in which mRNA is “read” by a ribosome, and amino acids, each attached to a tRNA bring the aa’s one at a time to the active site. • In vitro, protein synthesis can be done by a sequential process done on a machine. Peptide Bond is Planar * The peptide plane consists of six atoms, Ca1, C, O, N, H, Ca2 * H-N-Ca bond angle is 121o, not 109.5o. Resonance in Peptide Bond .. The peptide bond is rigid and planar due to the double bond resonance form, which shortens the C-N distance to 0.16 A shorter than a normal C-N bond. Properties of Peptide Bond • Barrier to Rotation about C-N bond is ~ 20 kcal • Peptide bond is planar with Ca groups mostly trans to the peptide bond (less steric clash of -R groups) • Ca groups are tetrahedral (3-D) and able to rotate though some angles are unfavorable due to steric repulsion with other atoms. • Ca are chiral are ALMOST exclusively the L stereoisomer. Peptides Conventional Nomenclature • N (amino) terminal on the left • C (carboxy) terminal on the right • biological synthesis of proteins on the ribosome occurs from N to C terminus. • Amino terminal amino acid is named residue 1. What are the thermodynamics of peptide bond formation? • G+A = G-A + H2O DGo = -RTlnK DGo = +5 kcal/mole DGo = -RT ln [Prod]/[Reac] – [Prod]/[React] = exp(-5000/RT) ≈ exp(-8) ≈ 3.3 x 10-4 – [Prod]/[React]= dipeptide/free amino acids ≈ 1/3000 • Only 1 in 3000 dipeptide bonds should remain intact at equilibrium in solution • Remaining peptide bonds should hydrolyze and exist as free amino acids! • Why don’t they? But first, why is hydrolysis favored thermodynamically? • Peptide Bond enthalpy doesn’t compensate for the increase in entropy gained by making two molecules rather than one molecule • Individual amino acids may also be better solvated. If hydrolysis is favored, why don't peptide bonds in the proteins in our body spontaneously undergo hydrolysis? Activation Barrier to Hydrolysis must be greater than kT Not only are peptide bonds created constantly in our bodies, the average lifetime of peptide bond is 100 years! • How are they made? – Thermodynamic strategy • Peptide bond formation is coupled to the breakdown of a high energy phosphate bond in a protein factory known as the ribosome. • How are they maintained? – Kinetic strategy • Enzymes which lower the activation barrier between product and reactant. For the hydrolysis of peptide bonds, these enzymes are called digestive enzymes. Peptide Backbone • three atoms from each amino acid on backbone – nitrogen, – alpha carbon, – carbonyl carbon. • other atoms of the polymer hang off of this chain. • This polymer has certain structural features which are invariant Summary of invariant backbone properties • • • • All amino acids have an L chirality at Ca Peptide bond itself is planar No rotation about peptide bond Ca on adjacent amino acids are most often trans to the peptide bond. – proline can be cis or trans Where does flexibility occur? • Variation in the 3-D structure of a protein is a result of rotational freedom about the sp3 hybridized Ca • Rotations of the backbone result in very different polymers and involve the relative orientation of two contiguous peptide planes What is a dihedral angle? • Dihedral angle is the angle – between four atoms • (Ha, C1, C2, Hd) – or across three bonds • (Ha-C1- C2- Hd) • It is measured by sighting down the central bond, or the central two atoms, and looking at the through space angle made by the atoms of the first bond relative to the atoms of the third bond. C1 C2 is eclipsed Dihedral angle in Ethane H3C-CH3 here is 60o Backbone Dihedral Angles f, Phi rotation: – the dihedral angle phi, f, represents the rotation about N-Ca – The four atoms which define f are the carbonyl carbonn-1, the nitrogenn, the a-carbonn, and the carbonyl carbonn. – Zero for f is where the two carbonyl carbons come into closest contact with one another. F Backbone Dihedral Angles y Psi rotation: – the dihedral angle psi, y., measures rotation about Ca-C – The four atoms which define y are the nitrogenn, the acarbonn, the carbonyl carbonn and the nitrogenn+1. – Zero for y is where the two nitrogens come into closest contact with one another. y Backbone Dihedral Angles • Rotations of f and y by definition are from the perspective of the chain direction (N to C terminal) – rotations are (+) clockwise; (-) counterclockwise • N-Ca for f • Ca-C for y • Stupid Pneumonic • Phi includes the n-1 residue, with the n amino acid saying to the n-1 amino acid, you're [p]history. • Psi includes the n+1 residue, with the n carbonyl [p]sighting ahead to the n+1 amino acid. Other degrees of Freedom • Rotations of the Side Groups – -R groups of the amino acids show a great deal of variability in non-backbone dihedrals – prolines • can only adopt certain phi,psi angles and so are not found in some types of secondary structure. • It is nearly impossible to twist a proline into an alpha helical orientation, thus it is known as a helix breaker. Molecular Architecture Secondary Structure • Ramachandran Plots are contour plots of the conformational energies as a function of psi (x-axis) and phi (y-axis) values for alpha carbons in protein backbones. RAMACHANDRAN GN, RAMAKRISHNAN C, SASISEKHARAN V., J Mol Biol., 7:95-99 • http://dicsoft1.physics.iisc.ernet.in/rp/ Beta Sheet Alpha helix Ramachandran plot Blue: helix Red: strand Green: turn and loop. Topological Lines outer, 90% probab. Inner, 50% probab. + non-gly aa X gly □ prolines. PDB NOSynthase Protein Structure Molecular Architecture • Phi and Psi Dihedrals 4 n+1 3,4 2,3 1,2 1 n-1 n – Phi rotation, f : the rotation of the trailing polypeptide about the Ca -N bond. The four atoms which define f are (1) the carbonyl carbonn-1, (2) the nitrogenn, (3) the a-carbonn, and (4) the carbonyl carbonn. – Psi rotation, y: the rotation of the leading polypeptide about the C"-C (carbonyl) bond. The four atoms which define y (1) the nitrogenn, (2) the alpha carbonn, (3) the carbonyl carbonn, and (4) the nitrogenn+1. Secondary Structure a-HELIX • a-HELIX, predicted by Pauling in 1948 and found experimentally by Kendrew in the crystal structure of myoglobin in 1953. – 3.613 right handed a-helix, which means that there are 3.6 residues per turn, and 13 atoms between hydrogen bonds. – 5.4 repeat unit, the length of one turn in an alpha helix. – 5.4/3.6 = 1.5 Å/residue. – dihedrals of approximately f -60o, y -50o Protein Structure a-HELIX • Favorable H-Bonding exists between the O of one amino acid residue and the N-H of a different amino acid residue four amino acids away. – A H bond is created when the electronegative lone pair electrons on the carbonyl oxygen cause the amide H to shift slightly away from N and towards O and form a weak bond. – O- - - H-N bond distance is ~2.9 Å – bond energy is ~ 10 kJ/mole or less (compared with 400kJ for a C-H bond). • Must remember that these atoms would be interacting with solvent if they weren’t involved in this internal H bonding. Protein Structure a-HELIX 13 atoms between hydrogen bonds. Protein Structure α-HELIX * In this highly idealized image of myoglobin, all of the helices are represented as colored ribbons, which trace their backbone. * For simplicity, all side groups are completely ignored. * How many helices do you see? See myoglobin file with Rasmol Protein Structure α-HELIX • Large Dipole Moment Along Helix Axis – is created by the alignment of the dipoles of each peptide along the helix axis. – is positive at the amino end and negative at the carboxy end of the helix. – 3.5 DeByes/peptide bond; 0.7 kcal/mole • Ref: Wada, Adv. Biophysics 9 1-63 (1976) – D or E stabilize helix when at N-terminus but destabilize helix what at C-terminus, • leads to depression in the pKa of D or E at N-term. – K or R destabilize at N-terminus, stabilize at Cterminus, • leads to a depression in the pKa of K or R at the Nterminus. Protein Structure α-HELIX Dipole Moments All Line Up Protein Structure α-HELIX Protein Structure α-HELIX • Physical Length: – Typical length of alpha αhelix is about 10 residues or 15 Å, remember 1.5 D /residue) though there's quite a range. Though it used to be thought that helices in proteins rarely got larger than 100 D – Now, images of proteins like tropomyosin at right reveal much larger helices that are helices of helices. See Tropomyosin ent files using Rasmol Protein Structure α-HELIX • -R groups big and bulky side groups accommodated – charged amino acids project away from the cylinder – CH2SH group of a C can combine with a -CH2SH side group of a second C to make a disulfide bond, -CH2S-SCH2-. – A, E, L and M are good alpha helix formers R groups on the outside of helix: – P, G, Y and S are poor helix formers Protein Structure α-HELIX • Human glucocorticoid receptor – pdbID 1M2Z glucocortisone dexamethasone