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CHMI 2227E Biochemistry I Peptides -General structure and properties CHMI 2227 - E.R. Gauthier, Ph.D. 1 Peptides Peptides are polymers of amino acids; Amino acids building blocks (residues) are linked to each other through a covalent bond: the peptide bond. 1 1 2 2 CHMI 2227 - E.R. Gauthier, Ph.D. A dipeptide 2 Peptides Polypeptides and proteins are simply chains of amino acids linked together through peptides bonds: If less than 20 residues: oligopeptide; If more than 20 residues but molecular mass (Mr) < 10,000 Da: polypeptides. If Mr > 10 kDa: protein. Particular terminology: Dipeptide (2 residues) / Tripeptide (3 residues) / Tetrapeptide (4 residues) / Pentapeptide (5 residues) / Ect, ect, ect. Note: 1 Da (dalton) = 1 g /mol. Little trick: Mr of a polypeptide/protein number amino acids x 110 Da. CHMI 2227 - ~ E.R. Gauthier,of Ph.D. 3 Peptides - polarity Each peptide has a polarity: One extremity with the NH2 (the one bonded to the Ca) which is not part of a peptide bond: N-terminal end; One extremity with the COOH (the one bonded to the Ca) which is not part of a peptide bond: C-terminal end; By convention: the N-terminal is always placed on the left, and the C-terminal on the right. CHMI 2227 - E.R. Gauthier, Ph.D. 4 Peptides - nomenclature Different ways to write this peptide (hyphen = peptide bond) : Tyrosyl-glycyl-glycyl-phenylalanyl-leucine Tyr-Gly-Gly-Phe-Leu Y-G-G-F-L YGGFL Note that the peptide is always written with the N-ter to the left and the C-ter to the right (NH2COOH). CHMI 2227 - E.R. Gauthier, Ph.D. 5 Peptide: hydrolysis The composition (NOT the sequence) of a peptide in its amino acid constituents is determined by first hydrolysing the peptide bond, and then identifying the amino acids: Tyr-Gly-Gly-Phe-Leu Gly2, Leu, Phe, Tyr 6 M HCl The amino acids are then purified by High Pressure Liquid Chromatography (HPLC). Detection is done by UV absorbance. To detect those amino acids that cannot absorb UV (you know which ones…), the amino acid are derivatized, meaning they are chemically coupled with a compound that absorbs UV. Quantification and identification of the amino acids is often done with the help of standards (analyzed with the same system but in a separate experiment); CHMI 2227 - E.R. Gauthier, Ph.D. 6 Analysis of amino acids Detection of amino acids: ninhydrin reagent O COOH OH OH C NH3+ R-HC=O CO2 O O R N O Ninhydrin 2 Amino acid O O Purple!! While Trp, Phe and Tyr can be detected by their A260-280nm, the other amino acids cannot; Ninhydrin reacts with the amine group of amino acids, generating a purple product (yellow in the case of Pro). The ninhydrin reaction allows one to detect and quantify (A570nm) the amino acids contained in the fractions of CHMI the IEX 2227column. - E.R. Gauthier, Ph.D. 7 High Pressure Liquid Chromatography (HPLC) PITC = phenylisothiocyanate PTC = phenylthiocarbamyl http://www.protein.iastate.edu/aaa.html CHMI 2227 - E.R. Gauthier, Ph.D. 8 High Pressure Liquid Chromatography (HPLC) http://www.protein.iastate.edu/aaa_figure3.html CHMI 2227 - E.R. Gauthier, Ph.D. The relative amount of each amino acid is given by calculating the area under each curve. 9 Peptide - ionization Each peptide will exist in different protonated forms, depending on the pH and its amino acid composition: Terminal amino and carboxyl groups can be protonated/ionized as in the free amino acid; The side chain can also be ionized, if an appropriate group is present; The NH2 and COOH groups that are part of the peptide bond are NOT ionized. Therefore, there will be a pH where a given peptide/protein will carry no net charges: this pH value will be the isoelectric point of the peptide/protein in question. Example: Ionization of the peptide GAVFD at pH 2, 6 and 12. CHMI 2227 - E.R. Gauthier, Ph.D. 10 Example of peptides 1. Aspartame: artificial sweetener COOCH2 O H3N+-CH-C-NH-CH-C-OCH3 CH2 O Asp-Phe-methyl ester 2. Oxytocin: stimulates uterine contractions Disulfide bond S S Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 Glycinamide residue: 2HN-CH2-CONH2 CHMI 2227 - E.R. Gauthier, Ph.D. 11 Example of peptides 3.Insulin Intrachain disulfide bond Interchain disulfide bonds CHMI 2227 - E.R. Gauthier, Ph.D. 12 Example of peptides 4.Cystic Fibrosis Transductance Regulator CHMI 2227 - E.R. Gauthier, Ph.D. Single polypeptide chain of 1480 amino acids; Responsible for the transport of chloride ions across the cell membrane; Mutation of F508 yields a non-functional protein and cystic fibrosis. What is the approximate Mr of CFTR? 13 General properties of proteins 1. Proteins differ in their Mr. Mr (kDa) Insulin 5.7 Cytochrome c 13 Ribonuclease A 13.7 Lysozyme 13.9 Myoglobin 16.9 Chymotrypsin 21.6 Chymotrypsinogen 22 Hemoglobin 64.5 Serum albumin 68.5 Hexokinase 102 Immunoglobulin G 145 RNA polymerase 450 Apolipoprotein B 513 Glutamate 1,000 dehydrogenase Protein # # chains residues 51 2 104 1 124 1 129 1 153 1 241 3 245 1 574 4 550 1 800 2 1,320 4 4,100 5 4,536 1 8,300 40 Source: Biochemistry. Lehninger. CHMI 2227 - E.R. Gauthier, Ph.D. 14 Multimeric proteins Proteins with more than one polypeptide chains are called multimeric proteins; Different types of multimeric proteins exist: Homo/oligomeric: 2 or more copies of the same polypeptide chain; Heteromeric: different polypeptide chains make up the proteins. The different polypeptides of a multimeric protein (i.e. the protein’s subunits) can be held together in different ways: Disulfide bonds Hydrogen bonds Hydrophobic interactions Electrostatic interactions Monomer/subunit 1 2 Heterodimer 1 1 Homodimer Hydrogen bonds: N-H ----- O-H N-H ----- N O-H ----- O=C N-H ----- O=C Electrostatic interactions: COO- ----- H3+N Multimeric proteins most often require all their parts in order to be functional. Very often proteins can change partners, providing them leading to their in/activation or giving them a different function. Hydrophobic interactions: -CH3 CH3CH3 CHMI 2227 - E.R. Gauthier, Ph.D. 15 Importance of multimeric proteins – planar cell polarity CHMI 2227 - E.R. Gauthier, Ph.D. 16 Importance of multimeric proteins – planar cell polarity Nature Genetics 38, 21 - 23 (2006) CHMI 2227 - E.R. Gauthier, Ph.D. 17 General properties of proteins 2. Proteins differ in their pI. Protein Pepsin pI 1 Egg albumin 4.6 Serum albumin 4.9 Urease 5 b-lactoglobulin 5.2 Hemoglobin 6.8 Myoglobin 7 Chymotrypsinogen 9.5 Cytochrome c 10.7 Lysozyme 11 Source: Biochemistry. Lehninger. CHMI 2227 - E.R. Gauthier, Ph.D. 18 General properties of proteins 3. Frequency of amino acid composition Number of residues per molecule of protein Amino Acid Human cytochrome c Bovine chymotrypsinogen Ala 6 22 Arg 2 4 Asn 5 15 Asp 3 8 Cys 2 10 Gln 2 10 Glu 8 5 Gly 13 23 His 3 2 Ile 8 10 Number of residues per molecule of protein Amino Acid Human cytochrome c Bovine chymotrypsinogen Leu 6 19 Lys 18 14 Met 3 2 Phe 3 6 Pro 4 9 Ser 2 28 Thr 7 23 Trp 1 8 Tyr 5 4 Val 3 23 Total 104 245 CHMI 2227 - E.R. Gauthier, Ph.D. 19 General properties of proteins 3. Frequency of amino acid composition CHMI 2227 - E.R. Gauthier, Ph.D. 20 General properties of proteins 4. Proteins can include other chemical groups in addition to amino acids Class Prosthetic group Example Lipoprotein Lipids b1-lipoprotein (blood) Glycoprotein Carbohydrates (sugars) Immunoglobulin G (blood) Phosphoprotein Phosphate groups Casein (milk) Hemoprotein Heme (iron porphyrin) Hemoglobin Flavoprotein Flavin nucleotides Succinate dehydrogenase Metalloprotein Fe Zn Ca Cu Ferritin Alcohol dehydrogenase Calmodulin Plastocyanin CHMI 2227 - E.R. Gauthier, Ph.D. 21 General properties of proteins 5. Proteins have a specific shape Each polypeptide spontaneously adopts a shape or conformation. A protein in its correct conformation is said to be native; This conformation is unique to each protein; Disruptions in the conformation (e.g. by heating) denatures the protein and usually leads to its inactivation. Globular proteins Fibrillar (rod-like) proteins CHMI 2227 - E.R. Gauthier, Ph.D. 22