Amino Acids, Peptides and Proteins - Handout | CHEM 501, Study notes of Chemistry

Download Amino Acids, Peptides and Proteins - Handout | CHEM 501 and more Study notes Chemistry in PDF only on Docsity! Chemistry 501 Handout 3 Amino Acids, Peptides, and Proteins Chapter 3 Dep. of Chemistry & Biochemistry Prof. Indig Lehninger. Principles of Biochemistry. by Nelson and Cox, 5th Edition; W.H. Freeman and Company Amino Acids Steric relationship of the stereoisomers of Alanine to the absolute configuration of L- and D-glyceraldehyde The amino acid residues in proteins are the L isomers Reversible formation of disulfide bond by the oxidation of two molecules of cysteine e.g. two polypeptide chains of insuline Aromatic R groups coo™ coo- CcOoO™ + | + | + HN—oH HIN—¢ —H H3;N—C —H CH, CH, CH, C=CH \ indole NH ring m \_/ Phenylalanine Tyrosine Tryptophan Absorption of ultraviolet light by aromatic amino acids Lambert-Beer Law log (Io/I) = ε C L Uncommon amino acids also have important functions Residues created by modification of common residues already incorporated into a polypeptide 12 34 5 αβγ plant cell wall, collagen collagen myosin prothrombim, a # of Ca+ binding proteins elastin Lysine residues ~ 300 additional amino acids have been found in cells rare, introduced during protein synthesis rather than created through a postsynthetic modification Reversible amino acid modifications involved in regulation of protein activity Titration of glycine Amino acids can act as acids and bases Nonionic and zwitterionic forms of amino acids amphoteric (ampholytes - amphoteric electrolytes) Titration curves predict the electric charge of amino acids Isoelectric point (or isoelectric pH) pI = ½ (pk1 + pk2) = ½ (2.34 + 9.60) = 5.97 TABLE 3-1 Properties and Conventions Associated with the Common Amino Acids Found in Proteins pK, values Abbreviation/ pK, pK, pK, Hydropathy Occurrence in Amino acid symbol M, (—COOh) (—NH} ) (R group) pl index* proteins (%)* Nonpolar, aliphatic R groups Glycine Gly G 75 2.34 9.60 5.97 —0.4 7.2 Alanine Ala A 89 2.34 9.69 6.01 1.8 7.8 Proline Pro P 115 1.99 10.96 6.48 1.6 52 Valine Val V 117 2.32 9.62 5.97 4.2 6.6 Leucine Leu L 131 2.36 9.60 5.98 3.8 9.1 Isoleucine lle | 131 2.36 9.68 6.02 4.5 5.3 Methionine Met M 149 2.28 9.21 5.74 1.9 2.3 Aromatic R groups Phenylalanine Phe F 165 1.83 9.13 5.48 2.8 3.9 Tyrosine Tyr Y 181 2.20 9.11 10.07 5.66 =1,3 32) Tryptophan Trp W 204 2.38 9.39 5.89 —0.9 1.4 *A scale combining hydrophobicity and hydrophilicity of R groups; it can be used to measure the tendency of an amino acid to seek an aqueous environment (— values) or a hy- drophobic environment (+ values). See Chapter 11. From Kyte, J. & Doolittle, R.F (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132. tAverage occurrence in more than 1,150 proteins. From Doolittle, R.F (1989) Redundancies in protein sequences. In Prediction of Protein Structure and the Principles of Protein Con- formation (Fasman, G.D., ed.), pp. 599-623, Plenum Press, New York. TABLE 3-1 Properties and Conventions Associated with the Common Amino Acids Found in Proteins pK, values Abbreviation/ pK, pK, pK, Hydropathy Occurrence in Amino acid symbol M, — (—COOH) (—NHF ) (R group) pl index* proteins (%)* Polar, uncharged R groups Serine Ser S 105 2.21 9.15 5.68 —0.8 6.8 Threonine Thr T 119 2.11 9.62 5.87 —0.7 5.9 Cysteine Cys C 121 1.96 10.28 8.18 5.07 2.5 1.9 Asparagine Asn N 132 2.02 8.80 5.41 35 43 Glutamine Gin Q 146 247 9.13 5.65 =3.5 4.2 Positively charged R groups Lysine Lys K 146 2.18 8.95 10.53 9.74 —3.9 5.9 Histidine His H 155 1.82 9.17 6.00 7.59 3.2 23 Arginine Arg R 174 2.17 9.04 12.48 10.76 —45 5.1 Negatively charged R groups Aspartate Asp D 133 1.88 9.60 3.65 27 3.5 5.3 Glutamate Glu E 147 2.19 9.67 4.25 3.22 —3.5 6.3 *A scale combining hydrophobicity and hydrophilicity of R groups; it can be used to measure the tendency of an amino acid to seek an aqueous environment (— values) or a hy- drophobic environment (+ values). See Chapter 11. From Kyte, J. & Doolittle, R.F (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132, TAverage occurrence in more than 1,150 proteins. From Doolittle, R.F. (1989) Redundancies in protein sequences. In Prediction of Protein Structure and the Principles of Protein Con- formation (Fasman, G.D., ed.), pp. 599-623, Plenum Press, New York. Peptides are chains of amino acids Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide Serylglyciltyrosylalanylleucine or Ser-Gly-Tyr-Ala-Leu or SGYAL Pentapeptide Peptides are named beginning with the amino-terminal residue, which by convention is placed at the left. condensationhydrolysis just a few residues → oligopeptide many residues → polypeptide Protein Separation and Purification Column Chromatography Crude extract --> --> --> fractionation lon-Exchange Chromatography @ Large net positive charge © Net positive charge © Net negative charge @ Large net negative charge Polymer beads with negatively charged functional groups Protein mixture is added to column containing cation exchangers. Example: Cation-exchange chromatography eal) 123456 Proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with a more negative net charge move faster and elute earlier. Exclusion Chromatography (gel filtration) polymer beads Protein mixture is added to column containing cross-linked polymer. Protein molecules separate by size; larger molecules pass more freely, appearing in the earlier fractions. Electrophoresis Cross-linked polymer polyacrylamide acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. SDS-polyacrylamide gel SDS CH3(CH2)11SO4-Na+ Purification of RNA polymerize from E. coli gel stained with a protein-specific dye (e.g. coomasie blue) Isoelectric focusing ‘An ampholyte solution is incorporated intoa gel. pHO. ane Decreasing pH pH3 | @ Astable pH gradient Protein solution is After staining, proteins isestablished inthe added and electric gelafter application _fieldis reapplied. of an electric field. are shown to be distributed along pH gradient according to their pl values. TABLE 3-6 The Isoelectric Points of Some Proteins Protein Pepsin Egg albumin Serum albumin Urease B-Lactoglobulin Hemoglobin Myoglobin Chymotrypsinogen Cytochrome c Lysozyme pl <1.0 46 4.9 5.0 5.2 6.8 7.0 9.5 10.7 11.0 Two-dimensional electrophoresis .___ First Decreasing dimension pl Isoelectric focusing | ®)) )) ))) Isoelectric focusing gel is placed on SDS. polyacrylamide gel. Isoelectric focusing [en D9. DIF gelis placed on SDS polyacrylamide gel. Second dimension SDS polyacrylamide gel electrophoresis There are several levels of protein structure Includes disulfide bonds Particularly stable arrangements of amino acid residues giving rise to recurring structural paterns All aspects of the 3-D folding of a polypeptide Multisubunit proteins Arrangement is space of polypeptide subunits Large proteins must be sequenced in smaller fragments Breaking disulfide bonds Cleaving the polypeptide chain Some proteases cleave only the peptide bond adjacent to particular amino acid residues Ordering the peptide fragments TABLE 3-7 The Specificity of Some Common Methods for Fragmenting Polypeptide Chains Reagent (biological source)* Cleavage pointst Trypsin Lys, Arg (C) (bovine pancreas) Submaxillarus protease arg (C) (mouse submaxillary gland) Chymotrypsin Phe, Tp, Tyr (C) (bovine pancreas) Staphylococcus aureus V8 protease Asp, Glu (C) (bacterium S. aureus) Asp-N-protease Asp, Glu (N) (bacterium Pseudomonas fragi) Pepsin Phe, Tip, Tyr (N) (porcine stomach) Endoproteinase Lys C Lys (C) (bacterium Lysobacter enzymogenes) Cyanogen bromide Met (C) 5-S Polypeptide Procedure hydrolyze; separate amino acids ammons react with FDNB; hydrolyze; separate amino acids reduce disulfide bonds (if present) SH cleave with trypsin; separate fragments; sequence by Edman degradation @®@O@®@ cleave with cyanogen bromide; separate fragments; sequence by Edman degradation Met (C) ©OO© establish sequence @ Wwenenu Result H 2 [Ra] La s 2 T 1 2 vi Y 2 P 3 2,4-Dinitrophenylglutamate detected GASMALIK EGAAYHDFEPIDPR DCVHSD YLIACGPMTK EGAAYHDFEPIDPRGASM TKDCVHSD ALIKYLIACGPM © Conclusion Polypeptide has 38 amino acid residues. Tryp- sin will cleave three times (at one R (Arg) and two K (Lys)) to give four frag- ments. Cyanogen bromide will cleave at two M (Met) to give three fragments. E (Glu) is amino- terminal residue. 2) placed at amino terminus because it begins with E (Glu). placed at carboxyl terminus because it does not end with R (Arg) or K (Lys). © overlaps with (-)and€-4),allowing them to be ordered. Amino terminus |EGAAYHDFEPIDPRGASMALIKYLIACGPMTKDCVHSD. 1 Carboxyl terminus © eo @ Amino acid sequences can also be deduced by other methods Correspondence of DNA and amino acid sequences codon O R' O | | [ | c ote ON CMe Coe Ce H Amino acid Fmoc residue (9-fuorenylmethoxycarbonyl) Nonpolar, aliphatic R groups coo" coo" coo™ coo- + + | LH + | H3N—C—H =-H3N—C—H i H3;N—C—H | | HON CH2 | CH; Ab H.C CHa city ‘CHs Glycine Alanine Proline Valine coo- coo" coo- + + | + HAN —5-H HN—C—H H3;N—C—H Ci cma cts cH Ae i i CH3 ~CH3 cH; : CH Leucine Isoleucine = Methionine Aromatic R groups coo coo coo™ * «1 , H;N—C—H H;N—C—H HN—¢ —H CH, | | cH, cH, ¢=CH x O QO NH OH Phenylalanine Tyrosine Tryptophan Polar, uncharged R groups coo" coo" coo" H3;N—C—H H;N—C—H H,N—C—H CH,OH H—C—OH CHa CH3 hy Serine Threonine Cysteine Arginine Histidine coo- coo Wi—C—H w—c—H He oa ¢ CHa aN Hn’ So i 7X Hn’ Yo Asparagine Glutamine Negatively charged R groups coo- coo- + | + | H-t-H H3;N—C—H CH, a coo- CH, coo™ Aspartate Glutamate