Chapter 5: Amino Acids, Peptides & Proteins Proteins & Polypeptides Are .

Chapter 5: Amino Acids, Peptides & Proteins – Amino acids share common functional groups Amino, Carboxyl & H bonded to Cα – Distinct chemistry of αα’s result of side chains αα’s categorized on basis of R group (side chain) Some R groups can also ionize (HA – H A -) Solubility, size, shape, charge, binding patterns Protein structure is defined at 4 levels – αα’s can act as both weak acids & weak bases Peptides 100 residues, Proteins 100 residues Proteins are stud ied using variety of methods – Nonpolar, aromatic, polar, ( )-charged, (-)-charged – Proteins & polypeptides are polymers of αα’s Primary (1 ) Secondary (2 ) Tertiary (3 ) Quarternary (4 ) Proteins with similar functions often have similar αα sequences 1 Amino Acids - the alphabet of protein structure 2 Side Chain (“R group”) Examples α β γ δ Amino acids share common structural features F ig 5.5 4 3-letter and 1- letter amino acid abbre viations The alpha carbon is a chiral center 5 6 1

Amino Acid s are c lassified ac cor ding to their R grou p Stereoisomerism in amino acids α α α α F ig 5.5 7 Nonpolar, Aliphatic R groups 8 Polar, Uncharge d R groups Aromatic R groups Fig 5.5 F ig 5.5 9 Positively Charged (Basic) R groups 10 Negatively Charged (Acidic) R groups F ig 5.5 F ig 5.5 11 12 2

Nonstand ard a mino acid re sidues Amino acids can act as acids and bases Fig. 5.8 F ig . 5.9 Created by modification of residues within proteins 13 Amino Acids have characteristic titration curves Zwitterions - dipolar ions 14 Effects of chemical environment on pK a pK pK a a [RN H 3 ] [R NH 2] Isoelectric Isoelectricpoint point pK pK a a F ig 5.11 [RCOOH OOH] [ ] [RCO RCOOO- ]- ] [RC 15 F ig 5.10 αα’s have at least 2 regions of buffer ing power Isoelectric point calculations (pI) pK a [RN H 3 ] [R NH 2] (ex: R-COOH, R -NH3 ) 16 (1) determine the number of ionizable functional groups (-COOH, -NH3 , -OH, -SH, etc.) Isoeletric point (pI) pH Where net charge on αα 0 For α α’s with 2 ionizable Groups pKa ’s of f unctional groups are influenced by neighbor s (2) Draw the series of proton losses from low to high pH 2 Isoelectric point pK a (3)Deter mine which species has a net charge of 0 1 [RC OOH ] [ RCO O - ] pI pK1 pK 2 2.3 9.6 2 2 5.97 (4) Average the pK’s which bracket that ionization F ig 5.10 17 18 3

αα’s with ionizable R groups have more complex titration curves Histidine - Biological Proton donor/Acceptor 3 stage titration curve α-CO OH α-CO O- H δ-COOH δ-CO O- H α-NH3 α-NH2 H pI pK1 pK 2 2.19 4.25 2 2 3.22 F ig 5.12 a F ig 5.12 b G lu tamate 19 Only αα with R group pKa near physiological pH (7.4) 20 Peptides and Proteins Activation of -COOH by tRNA F ig 5.13 αα’s are cova lently joined throu gh amide linka ges 22 Peptide & Protein Terminology Biologically Active Peptides F ig . 5.14 Oligopeptide - few αα’s Polypeptid e - 20 αα’s Peptides, proteins have am ino termina l (N-termina l) and carboxyl-te rminal (C-termina l) ends Peptides & pr oteins a lso have Isoe lectric points (pI ’s) 23 Asapartame (Nutrasweet) 2 αα’s Oxytocin (uterine contractions) 9 αα’s Bradykinin (inflammation inhibitor) 9 αα’s Insulin (sugar uptake) 30/21αα’s Compare to proteins: Cytochrome C (energy metabolism) 104 αα’s Titin (muscle protein) 27000 αα’s 24 4

Proteins can have single or m ultiple polype ptide chains Multiple subunits Multimeric proteins Subunits can be same or differ ent – – Disulfide bonds Ex: actin (identical subunits) Ex: Hemoglobing (different subunits) Dimers, trimers, tetramers, etc. refer to number of subunits in multimeric proteins F ig . 5.7 Subunits held together by noncovalent and covalent (disulfide bonds) linkages 25 26 Polypeptides have characteristic αα compostion Chemical modification of proteins Hydrolysis yields chara cte ristic proportion of αα’s The stand ard 20 αα’s almost never occu r in equal proportions Proteins w ith d iff erent fu nctions w ill d iffe r significantly in their r espective pr oportions Crude yet power ful method of protein identification 27 28 Working with proteins Levels of structure in Proteins Proteins must be purified before they can be characterized – Knowledge of structure and fu nction Proteins are purified base d on their physical and chemical properties F ig 5.16 1 2 – 3 – 4 – 29 – Size (fractionation) Solubility (function of pH, temperature, salt) Charge (binding to oppositely charged compounds) Binding properties (ligands) 30 5

Unseparated proteins can be quantified by measuring catalytic activity Chromatography Takes advantage of differences in: Charge Size Binding affinity Mobile (liquid) and Stationary (matrix) phases (a) (b) S ame activity as (a), but higher sp ecific activ ity F ig 5.23 F ig 5.18 31 Proteins can be purified several thousand fold via combination of chromatography steps Activity total units of enzyme in solution Specific Activity number of units of enzyme per milligram of protein Electrophoresis: Seperation & Characterization Gel serves as a Molecula r “sie ve” F ig 5.19 Molecular weight 33 F ig 5.20 34 Covalent Structure of Proteins Electrophoresis - Isoelectric point (pI) Purified proteins – – – – Size (electrophoresis) Charge (electrophoresis or chromatography) Catalytic activity (enzyme assays) Binding properties (ligands) Detailed B iochemical analysis (Structure/Function) – – F ig 5.21 – 35 1 - amino acid sequence 2 - Circular dichroism 3 - X-ray crystallography, NMR 36 6

1 provides important Biochemical Information Insights into 3D structure – Determining the 1 (1) Purification of protein α-helix, β-sheet, etc (2) Brea k disulfide (-S-S-) bond s (3) Cleave protein into smaller peptide fragments Cellular location of protein – - Enzymatic digestion Cytosol, plasma membrane, nucleus, etc. (4) Chemical modification & hydrolysis Evolutionary relationships Genetic disease (5) Id entification of individual amino acids - Isoelectric point, etc. 37 38 1 provides information on structure & function Cellular location or chemical modification – – – 1 seq uence information (contd.) N-terminal sequences - signals for export to nucleus, etc. Ser, Thr, Tyr context - phosphorylation signals Asn, Ser, Thr context - glycosylation signals – – – – Functional similarity Evolutionary relationship Sickle cell anemia, Cystic Fibrosis, MD Comparison to healthy individuals Single amino acid changes in many cases – – Homologous proteins share a significant amount of sequence identity ( 25%) Evolutionarily related Usually perform the same function in different species Bo x 5 .2 F ig 1 Most proteins are polymorphic - exhibit variation in αα sequence within species – 41 cytochrome C Gradation in variation: – 40 Positions which vary in αα sequence are c alled variable residues Protein homology & Polymorphism Genetic disease – 39 Catalytic sites: KTGG L (glucose pocket) LxxxxxxLxxxxxxL : “leucine zipper” NNRKN (Basic ( ) residues): DNA binding domain Etc. – Comparison to sequences w/known structure – Structural domains Conservative (hydrophobic for hydrophobic αα) Nonconservative (Polar for hydrophobic, etc.) 42 7

Biomedical & related benefits Evolutionary relationships Forensic Science – Identification of individuals, populations – Proteomics – – – Examination of the expression of all proteins in a cell Comparison of healthy/diseased states - what proteins are (not) expressed? Provide targets for drug development – – – – – 43 Nonpolar aliphatic Aromatic Polar uncharged Acidic (-) charged Basic ( ) charged 44 αα’s ionize in aqueous solution – – α-COOH α-COO α-NH3 α-NH2 H R Group ionization Proteins are often conjugated to other molecules – H – – – 45 Amino (N-) and Carboxy (C-) terminal ends Metal ions Lipids Carbohydrates Etc. 46 Primary, secondary, tertiary & quarternary Protein structural & functional analysis – – – αα’s are covalently joined through peptide bonds Summary (contd.) 4 levels of protein structure – Summary (contd.) αα’s are often characterized by their isoelectric point (pI) - pH where they have no net charge – - 2 stereoisomeric forms (D- and L -) Only L- form in proteins – – α-car bon (central carbon) is asymmetric in all αα’s (exce pt glycine) Summary (contd.) αα’s classified on the basis of polarity & charge – α-COOH group α-NH2 group Distinctive side chain (R group) – – 20 Standard αα’s found in proteins Anthropology – Chapter 5 - Summary Solubility (Precipitation with salts) Chromatography (Size, Charge, Binding affinity) Electrophoresis (Size and charge) 1 provides important Biochemical Information – – 3D structure, active sites, targeting signals, etc. Protein homology and polymorphism 47 8

1 1 Chapter 5: Amino Acids, Peptides & Proteins Amino acids share common functional groups - Amino, Carboxyl & H bonded to Cα Distinct chemistry of αα's result of side chains αα's categorized on basis of R group (side chain) - Nonpolar, aromatic, polar, ( )-charged, (-)-charged αα's can act as both weak acids & weak bases - Some R groups can also ionize (HA .