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Transcript
Amino Acids and Proteins Muhammad Jawad Hassan Assistant Professor Biochemistry Objectives • Structure and Classification of amino acids • Peptide Bond and Primary structure of protein • Secondary Structure of protein, Helices and Sheets • Tertiary and Quaternary Structure of protein, domain and motifs • Structure-function relationship of proteins and disease Structure dictates function Protein structure allows DNA replication without dissociation of replicating machinery 3 Protein subunits: amino acids: L & D isomers Only L amino acids found in proteins. C chiral, L & D isomers not symmetrical, except glycine R group = side chains Amino group Carboxylic acid group Mirror images of each other The 20 Amino Acids The amino acids each have their own shape and charge due to their specific R group. View the molecular shape of amino acids by clicking on the URL link below: http://sosnick.uchicago.edu/amino_acids.html Would the shape of a protein be affected if the wrong amino acid were added to a growing protein chain? Ionization state as a function of pH Physiological pH (measure of [H+]) Simplest amino acids Ball & stick Stereochemical Fischer projections Aliphatic side chains M: thioether (-S-) Ile: 2nd chiral center Aliphatic side chains hydrophobic Proline: cyclic structure Ring structure: Proline conformationally restricted, marked effect on protein architecture Aromatic side chains Cysteine Similar to Serine with sulfhydryl, or thiol (-SH) group replacing hydroxyl (-OH) group -SH more reactive than -OH. -SH pairs form disulfide bonds (aka bridges), key role stabilizing proteins The basic amino acids Polar side chains Lys & Arg have positive charges at neutral pH Lys side chain capped with amino group His can be positively charged near physiological pH Carboxylate & Carboxamide side chains pKa of some amino acids Amino acid abbreviations Essential Amino Acids • 10 amino acids not synthesized by the body • arg, his, ile, leu, lys, met, phe, thr, trp, val • Must obtain from the diet • All in diary products • 1 or more missing in grains and vegetables 16 Primary structure: Peptide bond, between AAs Between -carboxyl group of one AA & -amino group of another 2 amino acids Dipeptide Equilibrium favors hydrolysis, hence, biosynthesis of peptide bonds require free energy input Peptide bonds are stable kinetically Loss of H2O Polypeptide chain has direction Main chain or backbone Constant backbone: regularly repeating part Distinctive side chains (R-groups): variable part AA unit in a polypeptide is called a residue, which contains, a carbonyl group; good hydrogen-bond acceptor, an NH group (except Pro); good hydrogen-bond donor Cross links (disulfide bridges) Prevalent mainly in extracellular proteins Bovine insulin: AA sequence 1953, Fred Sanger determined aa sequence of insulin, landmark! Showed for 1st time, protein has precisely defined aa sequence Also showed that only L-amino acids were present, linked by peptide bonds Now, aa sequence of > 100,000 proteins are known 1950s-1960s studies showed aa sequence genetically determined Each of 20 aa encoded by one or more specific sequences of 3 nucleotides. Polypeptide bonds are planar Six atoms (Ca, C, O, N, H, Ca) lie in a plane, in a pair of aa Bond lengths in peptide unit Trans & cis peptides Cis configuration has steric hindrance; trans strongly favored Rotation of bonds in a polypeptide Amino group to C & carbonyl group to C are pure single bonds, allow rotation Freedom of rotation allows proteins to fold in different ways Dihedral angle: measure of rotation about a bond between -180o & +180o Ramachandran diagram Most angle combinations (75%) excluded by steric hindrance Dark green most favored Steric exclusion: powerful organizing principle Limited conformations favor protein folding, favorable entropy of too many conformations opposes folding Secondary structure: (1) alpha helix 1951, predicted by Pauling & Corey, 6 years before it was seen! ball & stick, side ribbon end view space-filling core alpha helix stabilized by hydrogen bonds CO group of residue n forms H-bond with NH group of Residue n + 4 Ball & stick model of alpha helix Ribbon and cylindrical depiction Residues related to each other by a rise of 1.5 Å and a rotation of 100 degrees. 3.6 aa residues / turn Pitch = 5.4 Å(1.5x3.6) Ferritin, an iron storage protein 75% alpha helix Helical content of proteins ranges widely Super helix: alpha helical coiled coil Can be as long as 1000 Å, very stable Helical cables in these proteins serve a mechanical role, forming stiff bundles of fibers Found in: • myosin and tropomyosin in muscle, • fibrin in blood clots, • keratin in hair, quills, claws, hoofs, & horns • intermediate filaments (cytoskeleton or internal scaffolding of cells) Structure of a beta strand Also predicted by Pauling & Corey Side chains are alternately above and below plane of backbone Distance between adjacent aa = 3.5 A Contrast to 1.5 A for alpha helix Antiparallel beta sheet Strands linked by H-bonding between opposite amino acids Parallel beta sheet Strands linked by H-bonding of an aa on one strand to two different aa on the adjacent strand Structure of mixed beta sheet Fatty acid-binding protein Rich in beta sheets Arrow pointing to carboxylterminal end Tertiary structure, myoglobin O2 carrier in muscle, 1st protein in atomic detail, 153 aa, X-ray crystals Tertiary structure, myoglobin, schematic Mainly alpha helices, total = 8 helices (75% of main chain) Prosthetic (helper) group to bind O2 Heme group is protoporphyrin IX, & central iron atom Distribution of aa in myoglobin Surface, mainly charged aa. Yellow: hydrophobic aa Blue: charged aa White: other aa Interior, mainly hydrophobic aa Cross-section Quaternary structure, dimer Cro protein of bacteriophage lambda Dimer of identical subunits Quaternary structure, tetramer Human hemoglobin, two alpha(red) two beta(yellow) subunits, 4 heme groups Covalent bond…..NO Amino acid sequence determines 3D-structure Bovine ribonuclease, 1950, C. Anfinsen work Denature & renature 4 disulfide bonds 124 amino acids Primary structure determination •Acid hydrolysis •Column chromatography •Ion exchange chromatography Reducing disulfied bonds beta-mercaptoethanol, reduced oxidized Denaturing agent, urea Denaturing agent, guanidinium chloride Denaturing agent, beta mercaptoethanol Ribonuclease: reduction & denaturation Finishing touches: covalent modifications Proteins covalently modified to augment function Research Protein Discuss what you can learn about its structure, function and the organism it comes from using the skills you learned today and website resources. You can explore a number of proteins using Cn3D. Go to the following URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Structure