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  1. Lehninger Principles of Biochemistry, Fourth Edition, 2005 Chapter 3AMINO ACIDS, PEPTIDES, AND PROTEINS 中央研究院 生物化學研究所 曾湘文 博士 Mar. 06, 2007

  2. 台大莊榮輝老師網頁:

  3. 3.1 Amino Acids 3.2 Peptides and Proteins 3.3 Working with Proteins 3.4 The Covalent Structure of Proteins 3.5 Protein Sequences and Evolution

  4. Amino Acids share common structural Features • Proteins are polymers of amino acids, with each amino acid residuejoined to its neighbor by a specific type of covalent bond. • The a-carbon of AA is a chiral center. Molecules with a chiral center are optically active, they rotate plane-polarized light. • The additional carbons in an R group are designated b, g, d, e etc. • Carbon atoms are numbered from one end, giving priority to carbons with substitutions. containing atoms with the highest atomic numbers.

  5. D, L System (levorotatory vs. dextrorotatory) • Enantiomers -nonsuperimposable mirror images of each other the two forms represent a class of stereoisomers. • The absolute configurations of simple sugars and amino acids are specified by the D, L system. • RS system is used in the systematic nomenclature of organic chemistry and describes more precisely the configuration of molecules with more than one chiral center. • Nearly all biological compounds with a chiral center occur naturally in only one stereoisomeric form, either D or L. The amino acid residues in protein molecules are exclusively L-stereoisomers. D-Amino acid residues have been found only in a few, generally small peptides, including some peptides of bacterial cell walls and certain peptide antibiotics.

  6. The R groups in this class of amino acids are nonpolar and hydrophobic.

  7. Glycine has the simplest structure, its very small side chain makes no real contribution to hydrophobic interactions Methionine, one of two sulfur containing amino acids. has a nonpolar thioether group. First AA residue in translation of proteins Alanine, Valine, Leucine, and Isoleucine could contribute to hydrophobic interaction. The secondary amino (imino) group of Pro is held in a rigid conformation that reduces the structural flexibility of polypeptide regions containing proline. Nonpolar, Alipatic R Groups

  8. Absorbance of UV by Aromatic Amino acids • All are relatively nonpolar (hydrophobic). • -OH group of throsine can form hydrogen bonds and are important functional group. Can be phosphorylated as well. • All can absorb UV light (280 nm), Tyrosine and Tryptophan are stronger than phenylalanine. Use for protein quantification.

  9. Polar, Uncharged R Groups • The R groups of these amino acids are more soluble in water, or more hydrophilic, than those of the nonpolar amino acids, because they contain functional groups that form hydrogen bonds with water. • Serine and Threonine has –OH, which contribute to polarity, and could be phosophorelated. • Asparagine and Glutamine are the amides of Aspartate, and Glutamate, and are easily hydrolyzed by acid or base.

  10. Reversible Formation of Disulfide Bond • Cysteine is readily oxidized to form a covalently linked dimeric AA called cystine (disulfide bond). • The disulfide-linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming covalent links between parts of a protein molecule or between two different polypeptide chains.

  11. Positively Charged (Basic) R Groups

  12. Positively Charged (Basic) R Groups • Lysine has a second primary amino group at e position. Its R group has significant positive at pH=7. • Arginine has a positively charged guanidino group • Histidine a imidazole group, and is the only standard amino acid having an ionizable side chain with a pKa near neutrality. It serves as a proton donor/ acceptor in a enzyme-catalyzed reaction

  13. Negatively Charged (Acidic) R Groups • Two amino acids having R groups with a net negative charge at pH=7 are asparate and glutamate, each of which has a second carboxyl group

  14. Classify the amino acids by polarity Juang RH (2003) Biochemistry

  15. Uncommon Amino Acids - I

  16. Nonstandard Amino Acids • Some 300 additional amino acids have been found in cells. • Are created by modification of standard residues already incorporated into a peptide. • 4-hydroxyproline, a derivative of proline, is found in plant cell wall protein and collagen; 5-hydroxylysine, derived from lysine, are found in collagen. • 6-N Methyllysine is a constituent of myosin. • g-Carboxyglutamate, found in the blood-clotting protein prothrombin and Ca2+ binding protein. • Desmosine, derivative of four Lys residues, which is found in the elastin. • Selenocysteine is introduced during protein synthesis, and contains Selenium rather than sulfur of cysteine, derived from serine.

  17. Uncommon Amino Acids - II • Ornithine and citrulline are not constituents of proteins. • They are key intermediates (metabolites) in the biosynthesis of arginine and in the urea cycle.

  18. Amino acids Can Act as Acids and Bases • Zwitterion (hybrid ion): dipolar ion, can act as either an acid (proton donor or a base (proton acceptor) - Amphoteric mater: ampholyte (amphoteric electrolytes)

  19. Amino Acids Have Characteristic Titration Curves • The pKa is a measure of the tendency of a group to give up a proton, with the tendency decreasing tenfold as the pKa increases by on unit.

  20. Titration Curves Predict the Electric Charge of Amino Acids • Isoelectric point (isoelectric pH): pI, The characteristic pH at which the net electric charge is zero (eg. glycine).

  21. Titration Curves of Glutamate

  22. Titration Curves of Histidine

  23. Effect of the chemical environment on pKa

  24. Effect of the chemical environment on pKa • The perturbed pKa of glycine is caused by repulsion between the departing proton and the nearby positively charged amino group. The opposite charges on the resulting zwitterion are stabilizing, nudging the equilibrium farther to the right. • The electronegative oxygen atoms in the carboxyl groups, which tend to pull electrons toward them, increasing the amino group to give up a proton.

  25. 3.2 Peptides and Proteins Formation of a Peptide Bond by Condensation

  26. The pentapeptide serylglycyltyrosylalanylleucine (Ser–Gly–Tyr–Ala–Leu) • A few amino acids are joined - an oligopeptide. • Many amino acids are joined, - a polypeptide. • “Protein” and “polypeptide” are sometimes used interchangeably, molecules referred to as polypeptides generally have molecular weights below 10,000 (D), and those called proteins have higher molecular weights. C terminal N terminal

  27. Peptides Can Be Distinguished by Their Ionization Behavior (Alanyl-glutamyl-glycyl-lysine) • The acid-base behavior of a peptide can be predicted from its free -amino and -carboxyl groups as well as the nature and number of its ionizable R groups. • Peptides have characteristic titration curves and a characteristic isoelectric pH (pI) at which they do not move in an electric field.

  28. Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes • Titin, a constituent of vertebrate muscle, which has 27,000 AAs, and M.W.=3,000,000. • Single peptide chain Vs. multisubunit protein: two or more polypeptide associated noncovalently. • The individual polypeptide chains in a multisubunit • protein may be identical or different. If at least two • are identical the protein is said to be oligomeric, and the identical units (consisting of one or more polypeptide chains) are referred to as protomers. • Ex. Hemoglobin- has four polypeptide subunits: two identical achains and two identical bchains, all four held together by noncovalent interactions. Each subunit is paired in an identical way with asubunit within the structure of this multisubunit protein, so that hemoglobin can be considered either a tetramer of four polypeptide subunits or a dimer of ab protomers. • The average M.W. of AA= 110 (128-18) NutraSweet - artificial sweetener

  29. Levels of Structure in Protein • Primary: A description of all covalent bonds. The sequence of AA residues • Secondary: particularly stable arrangements of AA giving rise to recurring structural patterns. • Tertiary: All aspects of the 3D folding of a polypeptide. • Quaternary: The spatial arrangement of multisubunits protein

  30. 3.3 Working with Proteins

  31. Separation and Purification of Proteins • Including size, charge, and binding properties. • Crude extract: breaking cells, by osmosis lysis or homogenization. • Fractionation: separate proteins into different fraction based on size of charge. • Salting out: The solubility of proteins is lowered at high salt concentration. Ammonium sulfate ((NH4)2SO4). • Dialysis is a procedure to separate proteins from solvents

  32. A Purification Table for a Hypothetical Enzyme 1. Crude cellular extract 2. Precipitation with ammonium sulfate 3. Ion-exchange chromatography 4. Size-exclusion chromatography 5. Affinity chromatography • Fraction volume (ml) • Total protein (mg) • Activity (units) • Specific activity (units/mg)

  33. The expansion of the protein band in the mobile phase is caused by separation of proteins with different properties and by diffusion spreading. As the length of the column increases, the resolution of two types of protein improves. Rate is decreased and resolution can decline because of the diffusion spreading. HPLC, or high-performance liquid chromatography. uses high-pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can withstand the crushing force of the pressurized flow. By reducing the transit time on the column, - limit diffusional spreading of protein bands and thus greatly improve resolution. Protein Purification: Column Chromatography

  34. Ion-exchange Chromatography (net electric charges) • The column matrix is a synthetic polymer containing bound charged groups; those with bound anionic groups (negatively charged) are called cation exchangers, • bound cationic groups (positively charged) are called anion exchangers. • Is effected by pH and salt concentration.

  35. Size-Exclusion Chromatography (size) • Also called gel filtration chromatography • The column matrix is a cross-linked polymer with pores of selected size. • Larger protein migrate faster than smaller ones because they are too large to enter the pores

  36. From: 台大莊榮輝上課資料

  37. Affinity Chromatography (binding specificity) • separates proteins by binding specificities. • The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads. • After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted by a solution containing free ligand.

  38. 9060 Polychrom (Diode Array) Detector Computer Workstation 9050 Variable UV/Vis Detector 9010 Solvent Delivery System HPLC Solvent Reservoirs Rheodyne Injector Keep an eye on these 4 screens! HPLC Column High Performance Liquid Chromatography (HPLC) use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.

  39. Normal vs. Reversed Phase Chromatography

  40. Electrophoresis • Separation of proteins is based on the migration of charged protein in an electric field • The migration of a protein in a gel during electrophoresis is a function of its size and shape. m = V / E = Z / f m :The electrophoretic mobility V: velocity; E: electrical potential Z: net charge f: frictional coefficient (shope)

  41. SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis • SDS binds to most proteins probably by hydrophobic interaction. One SDS for every two AAs, Thus, each protein has a similar charge-to-mass ratio. • Stains protein: Coomassie blue, Silver, and Sypro Ruby • Western blot

  42. Estimating the Molecular Weight of a Protein

  43. Isoelectric Focusing • pI of a protein: net charge=0 • A pH gradient is established by allowing a mixture of organic acids and bases (ampholytes). Protein migrates until it reaches the pH that matches its pI

  44. Two-Dimensional Electrophoresis • Separates proteins of identical MW that differ in pI or proteins with similar pI but different MW.

  45. First dimension: IEF (based on isoelectric point) - + Sample acidic basic High MW SDS-PAGE (based on molecular weight) Low MW Two-dimensional Gel Electrophoresis