1 thb_amino acids

Amino Acids: 

Amino Acids

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Amino acids – Amino acids are the fundamental building blocks of peptides and proteins. Amino acids are bonded by peptide bonds to form organizational units based upon the number of amino acids and structural shape. • Peptides – The peptides are molecules formed from amino acids that exist as: dipeptides (which contain two amino acids), tripeptides (which contain three amino acids, and polypeptides (which contain many amino acids). • Proteins – Proteins are molecules formed from coils (alpha-helices) of chains of amino acids. In addition to amino acids, proteins may contain other groups (such as the heme group of hemoglobin). • Peptides and proteins typically exist as molecules with complex structural organization. Hydrogen bonds and disulfide bonds form between peptide chains or different parts of a peptide chain, and are essential in provided the specific structure for the peptide or protein.

Proteins: Polymers of Amino Acids: 

Proteins: Polymers of Amino Acids 20 different amino acids: many combinations Proteins are made in the RIBOSOME

Amino Acid Chemistry: 

Amino Acid Chemistry NH 2 C a R 1 CO H NH C a R 2 COOH H NH 2 C a R COOH H amino acid 20 different types Amino acid Polypeptide Protein NH 2 C a R 1 COOH H NH 2 C a R 2 COOH H +

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Amino Acid Chemistry Discovered in 1806 (aspargine) 1938 (Threonine) Named from the source it is isolated Eg.: Aspargine – Asparagus Glutamate – Wheat gluten Tyrosine – Cheese (tyros – cheese) Glycine – Sweet taste (glykos – sweet)

Amino Acid Chemistry: 

Amino Acid Chemistry NH 2 C a R COOH H amino acid All amino acids have a central carbon (C , asymmetric) that is covalently bonded at four sites to the following: an amino group, NH2, an organic acid group, COOH, (3) a single hydrogen atom (H), and (4) a side chain called the “R” group. Exist in two forms which are nonsuperimposable images called enantiomers / stereoisomers Optically active (D & L forms) All amino acids differ in R group (w.r.t. structure, size, electric charge and solubility) Additional C in R is designated as , , ,  All 20 amino acids are standard / primary / normal / essential CORN

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Classification Based upon Shape, Size, H-bonding capacity and chemical reactivity Non Polar Aliphatic R-Groups Glycine Valine Alanine Leucine Proline Isoleucine

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Aromatic R-Groups Phenylalanine Tyrosine Tryptophan

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Polar, Uncharged R-Groups Serine Threonine Cystine Methionine Aspargine Glutamine

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Positively charge R-Groups Negatively charge R-Groups Histidine Lysine Arginine Glutamate Aspartate

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Ile Leu Lys Met Phe Pro Ser Thr Trp*** Tyr Val Ala Arg Asn Asp Cys Gln Glu Gly His What amino acids really look like

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Zwitter ions are electrically neutral and remain stationary in an electric field + NH 3 C a R COO - H Ionization of Amino Acids Easily ionized Can act as acids and bases Helps in understanding physical and biological properties of proteins (seperation, identification, quantification, sequencing etc) Amino acids crystallize from neutral aqueous solution as fully ionized species - Zwitter ion (German- hybrid) NH 2 C a R COOH H Nonionic form Zwitter ion form

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COOH COO - pKa ~ 2.2 NH 2 NH 3 + pKa ~ 9.4 The free amino and carboxylic acid groups have pKa’s Ionization of amino acids varies with pH + NH 3 C a R COO - H + NH 3 C a R COOH H NH 2 C a R COO - H - H + - H + + H + + H + pH = 1 pH = 7 pH = 11

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Amino acid Mol. Wt. pK1 pK2 Pk3 PI Hydropathy index Occ. in proteins (%) Glycine Gly G 75 2.34 9.60 - 5.97 -0.4 7.5 Alanine Ala A 89 2.34 9.69 - 6.01 1.8 9.0 Valine Val V 117 2.32 9.62 - 5.97 4.2 6.9 Leucine Leu L 131 2.36 9.60 - 5.98 3.8 7.5 Isoleucine Ile I 131 2.36 9.68 - 6.02 4.5 4.6 Proline Pro P 115 1.99 10.96 - 6.48 -1.6 4.6 Phenylalanine Phe F 165 1.83 9.13 - 5.48 2.8 3.5 Tyrosine Tyr Y 181 2.20 9.11 10.07 5.66 -1.3 3.5 Tryptophan Trp W 204 2.38 9.39 - 5.89 -0.9 1.1 Serine Ser S 105 2.21 9.15 13.60 5.68 -0.8 7.1 Threonine Thr T 119 2.11 9.62 13.60 5.87 -0.7 6.0 Cysteine Cys C 121 1.96 8.18 10.28 5.07 2.5 2.8 Methionine Met M 149 2.28 9.21 - 5.74 1.9 1.7 Aspargine Asn N 132 2.02 8.80 - 5.41 -3.5 4.4 Glutamine Gln Q 146 2.17 9.13 - 5.65 -3.5 3.9 Aspartate Asp D 133 1.88 9.60 3.65 2.77 -3.5 5.5 Glutamate Glu E 147 2.19 9.67 4.25 3.22 -3.5 6.2 Lysine Lys K 146 2.18 8.95 10.53 9.74 -3.9 7.0 Arginine Arg R 174 2.17 9.04 12.48 10.76 -4.5 4.7 Histidine His H 155 1.82 9.17 6.00 7.59 -3.2 2.1

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Reactions of amino acids The -carboxyl and -amino group exhibit similar chemical reactivity. Side chain exhibit specific reaction depending on nature of functional group Carboxyl group reactions:

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Amino group reactions: Acylation Acid halides

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Detection & Quantification. Ninhydrin or triketohydrindene hydrate strong oxidizing agent causes oxidative deamination of  amino. Products are an aldehyde, CO 2 , ammonia & hydrindantin-a reduced derivative of ninhydrin. Ammonia with hydrindantin & another molecule of ninhydrin yield purple product -Ruhemans purple, quantified spectrophotometrically at 570nm. CO 2 evolved is diagnostic of presence of  amino acid.  Imino acid – proline & hydroxyproline yields bright yellow ninhydrin product – 440 nm. Used in forensic as fingerprint detection as amino acids are components of human skin secretions. Ninhydrin reaction

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Specific reactions of side chains Important to study degradation, sequencing and synthesis of peptides & proteins To identify functional group at active site or label proteins for further studies

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Collagen & gelatin proteins Thyroglobulin Muscle proteins Proteins in corn Proteins of blood clotting Bacteriorhodopsin Uncommon amino acids: occur rarely

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Amino acids not found in proteins: AAs and their derivatives Biochemically important Neurotransmitter produced by decarboxylation of glutamic acid Metabolic intermediate Metabolic intermediate Metabolic intermediate

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Neurotransmitter derived from tryptophan Component of Pantothenic acid - part of CoA Precurssor of arginine Metabolic intermediate Adrenaline, derived from tyrosine, an hormone Neurotransmitter derived decarboxylation of histidine Constituent of penicillin Amino acids not found in proteins:

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Polymers of amino acids Range in size Peptide: 2-3 amino acid residue Protein: thousands of amino acid residue Two amino acids joined covalently by dehydration or condensation reaction, the result is a dipeptide with substituted amide linkage (peptide bond) and a molecule of water. Peptides & Proteins H COO - H H N C a R2 H + NH 3 C a R1 C OH O + H + NH 3 C a R1 C O COO - H N C a R2 H H 2 O

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Equillibrium of this reaction lies on the side of hydrolysis rather than synthesis. Can form dipeptide, tripeptide … oligo … poly …. Protein Amino acid in peptide is called residue In a peptide, AA residue with free amino group called amino terminal and is taken as the beginning of a peptide (left). It ends with a AA residue containing carboxyl group called carboxyl terminal (right). Peptide bond is stable : as hydrolysis of peptide is exergonic and occurs slowly due to high activation energy. (Disulphide bond also stabilizes peptide/protein)

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H + NH 3 C a R1 C O COO - H N C a R5 H C O H N C a R2 H C O H N C a R3 H C O H N C a R4 H Amino terminal Carboxyl terminal Repeating unit (NCC) called the main chain Variable part are the side chains (R1,R2,R3…) Since amino acids are combined into proteins by bonding at the carboxyl and amino groups, and these sites are usually not available for chemical reactions, it is the “R” groups that determine the role of the amino acids in proteins. Natural polypeptide has 50-2000 AA residues Avg. Mol. Wt. of AA is 110 Mol. Wt. of polypeptide is 5500 – 220000 Mass of protein is expressed as Dalton 1 dalton = 1 atomic mass unit protein of 50,000 MW have mass of 50,000 dalton or 50 kD

The Peptide Bond: 

The Peptide Bond Pauling and Corey  carbons of adjacent amino acids are separated by 3 covalent bonds [C - C – N - C] C – N bond in a peptide is shorter than C – N bond in a simple amine. Atoms associated with bond are coplanar Indicates resonance or partial sharing of pair of electrons between carbonyl oxygen and amide nitrogen. Sets electric dipole, making O partial –ve and N partial +ve -C - N- O = - H Resonance structures -C = N + - O - - - H -C-- N- O  - - H  +

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Peptide plane is flat w angle ~180º Partial double-bond Peptide bond The four atoms [O,C,N,H] lie in a plane, such that carbonyl carbon and H of amide nitrogen are in trans position. C-N are unable to rotate freely, due to partial double bond character (1.32A 0 ). (C-N = 1.49A 0 ; C=N = 1.27A 0; 40% double bond character) Rotation permitted about the single bonds N-C  ( phi) and C-C ( psi) 0 0 = same plane (-180 0 - +180 0 )

Implications of Peptide Planes: 

Implications of Peptide Planes w angle varies little, f and  angles vary alot Many f /  combinations cause atoms to collide Each residue is sandwiched between two planes C a H R f  Peptide planes C a H R C a

Polypeptide Backbone: 

Polypeptide Backbone Backbone restricted  limited conformations Collisions with side chain groups further limit f /  combinations C a H R f  C a H R C a H R

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Ionization behaviour Have one  amino group and one  carboxyl group Have different ionization constant than individual AAs Ionization of R group of AA also contribute to acid base behaviour. Have characteristic titration curves and isoelectric pH (used in seperation of peptides and proteins) H + NH 3 C a R1 C O COOH H N C a R2 H H + NH 3 C a R1 C O COO H N C a R2 H Cationic form (pH<3) Isoelectric form (pH<3) H + NH 2 C a R1 C O COO H N C a R2 H Non ionic form (pH 11)

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Biological activity of polypeptides Many small poly and oligopeptides have important biological activity at even low concentration. eg.: Hormones in vertebrates (intercellular messengers) Hormone Insulin (2PP; 30AA and 21 AA) Glucagon 29 residues Corticotropin 39 residues Bradykininin- hormone like peptide (inhibits inflammatory reactions) Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Oxytocin – formed by posterior pituitory gland Cys-Tyr-Ile-Gln-Asn-Cys-Pro_Leu-Gly S-S

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Enkephalins – Brain peptides, affect the perception of pain Tyr-Gly-Gly-Phe-Met Tyr-Gly-Gly-Phe-Met Gamicidin S – an antibiotic produced by Bacillus brevis D-Phe L-Leu L-Orn L-Val L-Pro L-Pro L-Val L-Orn L-Leu D-Phe

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Green fluorescent protein Aquorea victoria – a jelly fish Contains GFP works with protein aequorin to provide defense mechanism When attacked aequorin produces blue light, captured by GFP – emits bright green flash which blinds or startles the attacker. Flouorescence of GFP is w/o a prosthetic group, it is result of reaction between Ser-Tyr-Gly to form pigment complex-chromophore. Autocatalytic.

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Architecture of protein molecules: Protein shape – Basis of shape and solubility – Fibrous, Globular & Membrane Fibrous: simple, regular linear structures, serve structural roles in cells, insoluble in water or in dilute salt solutions. Globular: roughly spherical shape, polypeptide chain compactly folded with hydrophobic amino acids in the interior and hydrophillic on the outside exposed to solvent, water; soluble in aqueous solutions, cytosolic enzymes. Membrane: found in association with membrane system, have hydrophobic amino acid side chain oriented outward, insoluble in aqueous solutions but solubilized in detergents, have fewer hydrophillic amino acids than cytosolic

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Biological functions of Proteins: Are the agents of biological functions –

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Enzymes: are the catalyst that accelerates the rates of biological reactions 10 16 times that of uncatalyzed, >3000 enzymes are known. Eg. Alcohol dehydeganse, phosphofructokinase. Regulatory proteins: do not perform any chemical transformation but regulate the ability of other proteins to carry physiological functions. Eg. Insulin (5.7 kD), somatotropin (21 kD) - regulte gene expression eg. Repressors (+ve / -ve) Transport proteins: function to transport of sp. Substances from one place to other. Eg. Haemoglobin, serum albumin Membrane transport proteins eg. Channels Storage proteins: a reserviour of an essential nutrient, nitrogen – a limiting factor of growth is obtained from the amino acids of proteins. Eg. Ovalbumin, Casein, zeins (Zea mays or maize), phaseolin (phaseolus vulgaris) Ferritin – binds to as many 4500 atoms of iron

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Contractile or Motile proteins: endows cell the capabilities for movement, cell division, muscle contraction and cell motility. They are filamentous or polymerize to form filaments. Eg. Actin & myosin – contractile system tubulin – major component of microtubules dynein & kinesin – motor proteins drive movement of vesicles, granules and organelles Structural proteins: function in creating and maintaining biological structures, rpovide strngth and protection to cells and tissues, monomers polymerize to generate long fibres. Eg. -keratins – insoluble make up hairs, horns & fingernails collagen – found in bone, connective tissue, tendons, cartilage and hide – forms inelestic fibrils of great strength. elastin – elastic property to ligament fibroin (keratin) – constitutent of cocoons (silk) and spider web collagen & proteoglycans – protein-polysaccharide complex that cushion and lubricate

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Scaffold proteins (Adapter): Play imp role in complex pathways of cellular response to hormones and growth factors, Have modular organization in which specific parts of the proteins structure recognize and bind certain structural elements in other protein through protein-protein interaction. Eg. SH2 modules bind proteins in which tyrosine residue has been phosphorylated on its phenolic-OH. SH3 modules bind to proteins having characteristic grouping of proline residues PH molecules bind to membranes PDZ bind to C-terminal amino acid of certain proteins

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Exotic proteins: Monellin – protein in African plant have sweet taste, used as artificial sweetner for human consumption. Resilin – have elastic proteperties, found in hinges of insect wings Glue protein – secreted by marine organisms like mussels, allowing them to attach firmly to hard surfaces All the above property of proteins is attained by using just 20 amino acids Protective or Exploitive proteins: active role in cell defense, protection or exploitation. Eg. Immunoglobulins or antibodies produced by lymphocytes thrombin and fibrinogen – prevent loss of blood antifreeze proteins – Arctic and Antarctic fishes ricin – toxic plant protein, toxins in snake, bees, bacteria ( diphtheria & cholera )

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Proteins with chemical groups other than amino acids: Proteins with only amino acids and no chemical group are termed as Simple proteins . Eg. Ribonuclease, actin etc. Proteins with other chemical constituent as an integral part of its structure are termed as Conjugate proteins. If the non protein part is crucial for the proetein function it is referred as prosthetic group. Glycoproteins: contain carbohydrate, destined for an extracellular location. Eg.: Fibronectin & Proteoglycans –component of extracellular matrix that surrounds the cell of tissue in animals. Immunoglobulin G – antibody – circulating free in blood plasma. Membrane proteins are glycosylated

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Lipoproteins: conjugated with lipids Eg.: Blood plasma lipoproteins - function in transport of lipids to sites of active membrane synthesis. High density lipoproteins (HDL) (  -lipoprotein) Low density lipoproteins (LDL) (  -lipoprotein) Nucleoproteins: play role in the storage and transmission of genetic information. Virus particles (TMV, HIV-I, Adenovirus) and even chromosomes are protein-nucleic acid complexes. Phosphoproteins: have phosphate groups esterified to the hydroxyls of seirne, threonine or tyrosine residues. Eg.: Casein (milk protein) contain many phosphates Glycogen phosphorylase regulated between active & inactive states due to presence of phosphate.

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Metalloproteins: are metal storage forms Eg.: ferritin or enzymes in which the metal atom participates in a catalytically important manner. Alcohol dehydrogenase, Cytochrome oxidase, Nitrogenase, Pyruvate carboxylase Hemoproteins: are subclass of metalloproteins because their prosthetic group is heme, the name given to iron protoporphyrin IX Cytochrome c, Catalase, Nitrate reductase Flavoproteins: Flavin is an essential substance for the activity of a number of important oxidoreductases Succinate dehydrogenase (FAD) NADH dehydrogenase (FMN) Sulfite reductase (FMN, FAD)

Hierarchy of Protein Structure: 

Hierarchy of Protein Structure 20 different amino acids: many combinations The order of amino acids: Protein sequence Primary Structure Local conformation, depends on sequence Secondary Structure Overall structure of the chain(s) in full 3D Tertiary/Quaternary Structure

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The primary structure of a protein: Determination of amino acid sequence of protein: 1953 – Frederick sanger – reported sequences of 2 PP of insulin. More than 10000 protein sequence is available

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Protein sequencing strategy If the protein contains more than one polypeptide chain, the chains are separated and purified. Interchain S-S (disulphide) cross bridges between cysteine residues in the polypeptide chain are cleaved. The amino acid composition of each polypeptide chain is determined. The N-terminal and C-terminal residues are identified. Each polypeptide is cleaved in smaller fragments, and the amino acid composition and sequence of each fragment are determined. Step 5 repeated, using different cleavage procedure to generate a different and therefore overlapping set of peptide fragments. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments. The positions of S-S cross bridges formed between cysteine residues are located.

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Separation of polypeptide chains Homomultimer – contain only one type of polypeptide Heteromultimer – made of different kinds of polypeptide Expose to extreme pH, 8M Urea, 6M Guanidium hydrochloride or high salt concentrations – disrupts polar interactions like hydrogen bond both within the protein molecule and the protein and aqueous solvent. Separated based on differences in size and or charges The S-S disulphide bond to be cleaved prior to dissociation.

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Cleavage of disulphide bridges Number of methods available Method should not allow original or new bond formation Performic acid – results in two equivalents of cysteine - side chains are ionised SO 3 - - electrostatic repulsion and altered chemistry prevents S-S recombination Sulfhydryl compounds: 2-mercaptoethanol reduces S-S to regenerate two cysteine-SH residues. (SH groups recombine within or other SH group to form S-S bond)  it is followed by treating with alkylating agents – iodoacetate, 3-bromopropylamine to modify SH group.

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End group analysis Identification of N- and C- terminal residues No of ends in the protein (more than one end group – multiple polypeptides). N-terminal Analysis: Edman degradation – allows sequential identification of series of residues from N-terminal PTH derivative of N terminal is identified by chromatographic techniques. Rest of the polypeptide is intact ready for next round. For easy recovery of PP C-terminal is coupled to insoluble matrix. Reveals the sequence of protein Automated sequentors – 50 cycles – 50 pmol – 100-200 residue PP

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C-terminal Analysis: Enzymatic approach is commonly used. Carboxypeptidases – cleaves PP from C-terminal in a succesive manner. Four types: Carboxypeptidase A – (bovine pancrease) hydrolyze C-terminal peptide bond of all residues except proline, arginine and lysine Carboxypeptidase B – (analogous enzyme from hog pancrease) effective only when Arg and Lys are the C-terminal. Carboxypeptidase C – (Citrus leaves) – act on any C-terminal residue Carboxypeptidase Y – (from Yeast) – act on any C-terminal residue. Nature of C-terminal amino acid determines the overall rate at which it is cleaved as enzyme relives residue successively

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Fragmentation of polypeptide chain : Enzyme or some specific chemicals. Trypsin: is specific in hydrolyzing only peptides bonds in which carbonyl function is by an Arg or Lys residue. Number of smaller peptides is equal to the number of Arg & Lys residues in the protein. B. Chymotrypsin: hydrolyze peptide bond formed by the carboxyl group of the aromatic amino acids. over time it also hydrolyzes amide bonds of amino acids other than aromatic. Both A & B generate two different sets of fragments whose sequences overlap

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Nonspecific Endopeptidases: endopeptitidases (proteases) thet cleave peptide bonds within the interior of a polypeptide chain. Clostripain – acts on Arg residues. Endopeptidase Lys C – cleaves only at Lys residues. Staphylococcal protease – act on acidic residues Asp, Glu. Pepsin, papain, subtilisin, thermolysin and elastase . Papain (papaya), bromelain (pineapple) – causes hydrolysis of gelatin. Cyanogen Bromide: Widely used Acts upon methionine Nucleophilic S reacts with CNBr yielding sulfonium ion that undergoes rapid intramolecular rearrangement to form a cyclic iminolactone. Water hydrolyzes lactone, cleaving PP and generating fragments having C-terminal homoserine lactone residue

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Sulphonium ion iminolactone

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Other methods: Hydroxylamine (NH 2 OH) - cleaves at aspargine-glycine bond at pH 9 - cleaves at aspartyl-prolyl bonds under mildly acidic conditions

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Cleavage products generated by these procedures must be isolated and individually analyzed with respect to amino acid composition and end group analysis of small, overlapping peptides. Peptide sequencing today is most commonly done by Edman degradation of relatively large peptides

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Sequence determination by mass spectrometry (MS): Exploit difference in the mass-to-charge (m/z) ratio of ionized atoms or molecules to separate them from each other. Used for determining chemical and structural information. The basic operation of mass spectrometer is to: Evaporate and ionize moleculae in a vacuum, creating gas-phase ions, Separate the ions in space and/or time based on their m/z ratios, and Measure the amount of ions with specific m/z ratios.

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Location of Disulphide cross bridges: Disulphide bond is not a part of primary structure. Info can be obtained if bonds are not broken during sequencing methods As these bonds are stable, linked fragment can be isolated and identified within the protein digest. Diagonal electrophoresis

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Sequence databases: Database of protein – Atlas of protein sequence and structure Amino acid sequence- nucleotide sequence PIR – Protein Identification Resource Protein Sequence Database Gen Bank – Genetic Sequence Data bank EMBL – European Molecular Biology Laboratory Data Library

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Sequencing by tandem mass spectrometry: Tandem MS (or MS/MS) allows sequencing of proteins by hooking two mass spectrometers in tandem. First spectrometer is used to separate oligopeptides from a protein digest and then to select in turn each of these oligopeptides for further analysis Selected oligopeptide is fragmented by collision with helium or argon gas, collected fragments analysed by second spectrometer. Fragmentation occurs primarily in the peptide bonds linking successive amino acids