* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 1 1 2 bez pyt lecture chemistryofaminoacids 7 fin
Gene expression wikipedia , lookup
Expression vector wikipedia , lookup
Fatty acid metabolism wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Magnesium transporter wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Interactome wikipedia , lookup
Point mutation wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Western blot wikipedia , lookup
Metalloprotein wikipedia , lookup
Peptide synthesis wikipedia , lookup
Genetic code wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Biosynthesis wikipedia , lookup
2016-11-14 Chemistry of amino acids, peptides and proteins Amino acids • Provide the monomer units from which the long polypeptide chains of proteins are synthesized Derived Amino Acids Derived Amino Acids: Derived and Incorporated in proteins: • Some amino acids are modified after protein synthesis such as hydroxy proline and hydroxy lysine which are important component of collagen. • Gamma carboxylation of glutamic acid residues of proteins is important for clotting process. Coagulation (also known as clotting) is the process by which blood changes from a liquid to a gel, forming a blood clot. 1 2016-11-14 Derived Amino Acids: Derived and Incorporated in proteins: 4 Derived Amino Acids Derived Amino Acids: Derived but not incorporated in tissue proteins: e.g.: Ornithine, Citrulline, Homocysteine No-protein amino acids. L-Ornithine and citrulline • natural amino acid not found in proteins, • play a role in the urea cycle Derived Amino Acids Derived but not incorporated in tissue proteins: 2 2016-11-14 Derived Amino Acids Derived but not incorporated in tissue proteins: Homocysteine is biosynthesized from methionine by the removal of its terminal Cε methyl group Non standard amino acids Seleno cysteine - cysteine analogue with a selenium- in place of the sulfur-containing thiol group. Selenocysteine is present in several enzymes. Amino acids L-amino acids and their derivatives participate in cellular functions as diverse as nerve transmission and the biosynthesis of porphyrins, purines, pyrimidines, and urea. 3 2016-11-14 • Niacin, Serotonin and melatonin are synthesized from Tryptophan (vitamin B3) Melanin, thyroid hormone, catecholamines are synthesized from Tyrosine Epinephrine (adrenaline) GABA (neurotransmitter) is synthesized from Glutamic acid γ-amino butyric acid (GABA) 4 2016-11-14 Nitric oxide, a smooth muscle relaxant is synthesized from Arginine. Aminoacids are precursors for haem, creatine and glutathione, porphyrins, purines and pyrimidines. haem 15 5 2016-11-14 Reactions of amino acids 1. 2. 3. 4. Reactions due to amino group Reactions due to carboxyl group Reactions due to side chain Reaction due to both amino and carboxyl groups Reactions due to amino group Oxidative deamination-α amino group is removed and corresponding α-keto acid is formed. α-keto acid produced is either converted to glucose or ketone bodies or is completely oxidized. Reactions due to amino group Transamination-Transfer of an α amino group from an amino acid to an α keto acid to form a new amino acid and a corresponding keto acid. aspartate + α-ketoglutarate ⇔ oxaloacetate + glutamate 6 2016-11-14 Reactions due to amino group Formation of carbamino compound • CO2 binds to α amino acid on the globin chain of hemoglobin to form carbamino hemoglobin • The reaction takes place at alkaline pH and serves as a mechanism for the transfer of Carbon dioxide from the tissues to the lungs by hemoglobin. Reactions due to carboxyl group 1) Decarboxylation- Amino acids undergo alpha decarboxylation to form corresponding amines. ExamplesGlutamic acid GABA Histidine Histamine Tyrosine Tyramine 2) Formation of amide linkage • Non α carboxyl group of an acidic amino acid reacts with ammonia by condensation reaction to form corresponding amides Aspartic acid Asparagine Glutamic acid Glutamine Reactions due to carboxyl group 1) Decarboxylation- Amino acids undergo alpha decarboxylation to form corresponding amines. ExamplesGlutamic acid GABA Histidine Histamine Tyrosine Tyramine GABA is an inhibitory neurotransmitter whose receptors lower muscle tone, promote relaxation, diminish anxiety, and stimulate digestion. Histamine is involved in many allergic reactions. Tyramine acts as a catecholamine releasing agent. 7 2016-11-14 Reactions due to side chains 1) Ester formation • OH containing amino acids e.g. serine, threonine can form esters with phosphoric acid in the formation of phosphoproteins. Phospho-serine Reactions due to side chains: Glycoproteins 1) Ester formation • OH group containing amino acid can also form: Glycosides – by forming O- glycosidic bond with carbohydrate residues. O-linkage: The oxygen atom in the side chain of serine or threonine amino acids is attached to the sugar Reactions due to side chains: Glycoproteins N-linkage: The nitrogen atom in the side chain of Asparagine is attached to the sugar. 8 2016-11-14 Reactions due to side chains 2) Reactions due to SH group (Formation of disulphide bonds) • Cysteine has a sulfhydryl group( SH) group and can form a disulphide (S-S) bond with another cysteine residue. • The dimer is called Cystine • Two cysteine residues can connect two polypeptide chains by the formation of interchain disulphide chains. Formation of disulphide bond 26 Reactions due to side chains 3) Transmethylation The methyl group of Methionine can be transferred after activation to an acceptor for the formation of important biological compounds. 9 2016-11-14 Reactions due to side chains 4) Reactions due to both amino & carboxyl groups Formation of peptide bond Peptide Bonds Link Amino Acids in Proteins • Peptide bond - linkage between amino acids is a secondary amide bond • Formed by condensation of the α-carboxyl of one amino acid with the α-amino of another amino acid (loss of H2O molecule) Ala-Ser 29 Peptides and Proteins 20 amino acids are commonly found in protein. These 20 amino acids are linked together through “peptide bond forming peptides and proteins (what’s the difference?). - The chains containing less than 50 amino acids are called “peptides”, while those containing greater than 50 amino acids are called “proteins”. 30 10 2016-11-14 Peptide bond formation: - Each polypeptide chain starts on the left side by free amino group of the first amino acid enter in chain formation . It is N- terminus. - Each polypeptide chain ends on the right side by free COOH group of the last amino acid and termed (C-terminus). 31 Peptides Peptides • Amino acids linked by amide (peptide) bonds Gly H2Nend Lys Phe Peptide bonds Arg Ser -COOH end Glycyllysylphenylalanylarginylserine 33 11 2016-11-14 Resonance structure of the peptide bond (a) Peptide bond shown as a C-N single bond (b) Peptide bond shown as a double bond (40%) (c) Actual structure is a hybrid of the two resonance forms. Electrons are delocalized over three atoms: O, C, N 34 Trans and cis conformations of a peptide group • Nearly all peptide groups in proteins are in the trans conformation 35 Examples on Peptides: Dipeptide ( 2 amino acids joined by one peptide bond): Example: Aspartame which acts as sweetening agent being used in replacement of cane sugar. It is composed of aspartic acid and phenylalanine. Cannot be intaken by people suffered from phenylketonuria (phenylalanine hydroxylase is completely or nearly completely deficient, and Phe isn’t metabolised to Tyr) 36 12 2016-11-14 Aspartame, an artificial sweetener • Aspartame is a dipeptide methyl ester (aspartylphenylalanine methyl ester) • About 200 times sweeter than table sugar • Used in diet drinks • Shouldn’t be used with hot solutions and decomposes during heating Asp-Phe-OCH3 37 Examples on Peptides: Tripeptides ( 3 amino acids linked by two peptide bonds). Example: GSH - glutathione which is formed from 3 amino acids: glutamic acid, cysteine and glycine. It protects against hemolysis of RBC (Red Blood Cell) by breaking H2O2 which causes cell damage. Glu-Cys-Gly 38 Examples on Peptides: Octapeptides: (8 amino acids) Examples: Two hormones; oxytocine and vasopressin (ADH) 39 13 2016-11-14 Examples on Peptides: Polypeptides: 10- 50 amino acids: e.g. Insulin hormone, Chain B and Chain A 40 There Are Four Levels of Protein Structure •Primary structure - amino acid linear sequence •Secondary structure – the type and the shape of the peptide chain, such as a-helices and b-sheets •Tertiary structure - describes the shape of the fully folded polypeptide chain in space •Quaternary structure - arrangement of two or more polypeptide chains into multisubunit molecule 41 Primary Structure of Proteins The particular sequence of amino acids that is the backbone of a peptide chain or protein • ca 42 14 2016-11-14 Protein structure: Primary structure: The primary structure of a protein is its unique sequence of amino acids. Lysozyme, an enzyme that attacks bacteria, consists of a polypeptide chain of 129 amino acids. 43 High orders of Protein structure A functional protein is not just a polypeptide chain, but one or more polypeptides precisely twisted, folded and coiled into a molecule of unique shape (conformation). This conformation is essential for some protein function 44 Secondary Structure • Results from hydrogen bond between hydrogen of –NH group of peptide bond and the –O of C=O (carbonyl oxygen) of another peptide bond. • Three-dimensional arrangement of amino acids in a form of α- or β-structured of peptide bonds • Looks like a coiled “telephone cord” (α α) or nearly fully extended polypeptide chain (β) 45 15 2016-11-14 Secondary Structure According to H-bonding there are two main forms of secondary structure: α-helix: is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one β-sheets 46 Secondary Structure β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment). 47 Secondary Structure Amino acids Hydrogen bond Alpha helix Β-Pleated sheet 48 16 2016-11-14 The α Helix Is a Common Protein Secondary Structure • The α helix is a common type of secondary structure in proteins. • It is the predominant structure in α-keratins. • In globular proteins, about one-fourth of all amino acid residues are found in α helices 49 Stereo view of right-handed a-helix • All side chains project outward from helix axis 50 Secondary Structure – Beta Pleated Sheet • Polypeptide chains are arranged side by side • Hydrogen bonds between chains • R groups of extend above and below the sheet 51 17 2016-11-14 Secondary Structure – Beta Pleated Sheet The adjacent polypeptide chains in a β pleated sheet can be either antiparallel (having the opposite amino-to-carboxyl orientation) or parallel (having the same amino-to-carboxyl polypeptide orientation). 52 β -Sheets (a) parallel, (b) antiparallel 53 Secondary Structure – Beta Pleated Sheet • Typical of fibrous proteins such as silk (produced by the larva of the silkworm moth, to make cocoons) is almost pure antiparallel beta pleated sheet. • Elements of beta pleated sheet are found in many protein domains. 54 18 2016-11-14 Tertiary Structure • Specific overall shape of a protein • Cross links between R groups of amino acids in chain disulfide –S–S– + ionic –COO– H3N– H bond C=O HO– hydrophobic –CH3 H3C– 55 Tertiary Structure Is determined by a variety of interactions (bond formation) among R groups and between R groups and the polypeptide backbone 56 Tertiary Structure The weak interactions include: Hydrogen bonds among polar side chains Ionic bonds between charged R groups (basic and acidic amino acids) Hydrophobic interactions among hydrophobic ( non polar) R groups. 57 19 2016-11-14 Strong covalent bonds include disulfide bridges, that form between the sulfhydryl groups (SH) of cysteine monomers, stabilize the structure. 58 Formation of cystine (disulfide bridge) 59 Tertiary Structure Amino acids Hydrogen bond Alpha helix Pleated sheet Polypeptide (single subunit of transthyretin) 60 20 2016-11-14 Quaternary Structure •Refers to the organization of subunits in a protein with multiple subunits (2 or more chains) •Subunits (may be identical or different) •Subunits are held together by many weak, noncovalent interactions (hydrophobic, electrostatic) 61 Quaternary Structure Quaternary structure: two or more polypeptide subunits held together by non-covalent interaction like H-bonds, ionic or hydrophobic interactions. 62 Quaternary Structure • Examples on protein having quaternary structure: – Collagen is a fibrous protein of three polypeptides (trimeric) that are supercoiled like a rope. • This provides the structural strength for their role in connective tissue. ⅓ of structure is glycine, 10% proline, 10% hydroxyproline and 1% hydroxylysine. 63 21 2016-11-14 Quaternary Structure • Examples on protein having quaternary structure: – Hemoglobin is a globular protein with four polypeptide chains (tetrameric) 64 Hemoglobin tetramer (a) Human oxyhemoglobin (b) Tetramer schematic 65 Quaternary Structure • Examples on protein having quaternary structure: – Insulin : two polypeptide chains (dimeric) 66 22 2016-11-14 67 Conjugated proteins On hydrolysis, give protein part and non protein part are subclassified into: 1- Phosphoproteins: These are proteins conjugated with phosphate group. Phosphorus is attached to OH group of serine or threonine. e.g. Casein of milk and vitellin of yolk. 68 2- Lipoproteins: These are proteins conjugated with lipids. Functions: help lipids to transport in blood Enter in cell membrane structure helping lipid soluble substances to pass through cell membranes. 3- Glycoproteins: proteins conjugated with sugar (carbohydrate) e.g. – Mucin - Some hormones such as erythropoeitin - present in cell membrane structure - blood groups. 4- Nucleoproteins: These are basic proteins ( e.g. histones) conjugated with nucleic acid (DNA or RNA). e.g. a- chromosomes: are proteins conjugated with DNA b- Ribosomes: are proteins conjugated with RNA 69 23 2016-11-14 5- Metalloproteins: These are proteins conjugated with metal like iron, copper, zinc. a- Iron-containing proteins: Iron may present in heme such as in - hemoglobin (Hb) - myoglobin (protein of skeletal muscles and cardiacmuscle), - cytochromes, - catalase, peroxidases (destroy H2O2) - tryptophan pyrrolase (desrtroy indole ring of tryptophan). Iron may be present in free state ( not in heme) as in: - Ferritin: Main store of iron in the body. ferritin is present in liver, spleen and bone marrow. - Hemosidrin: another iron store. - Transferrin: is the iron carrier protein in plasma. 70 b- Copper containing proteins: e.g. - Ceruloplasmin which oxidizes ferrous ions into ferric ions. - Oxidase enzymes such as cytochrome oxidase. c- Zn containing proteins: e.g. Insulin and carbonic anhydrase d- Mg containing proteins:e.g. Kinases and phosphatases. 6-Chromoproteins: These are proteins conjugated with pigment. e.g. - All proteins containing heme (Hb, myoglobin) - Melanoprotein: e.g proteins of hair which contain melanin. 71 Protein Hydrolysis • • • • • Break down of peptide bonds Requires acid or base, or enzymes, water and heat Gives smaller peptides and amino acids Similar to digestion of proteins using enzymes Occurs in cells to provide amino acids to synthesize other proteins and tissues 72 24 2016-11-14 Hydrolysis of a Dipeptide OH + CH 3 O CH 2 O H 2O, H + H 3N CH C N CH C OH heat H OH CH 3 O + H 3N CH COH CH 2 O + + H 3N CH C OH 73 Denaturation - irreversible coagulation Disruption of secondary, tertiary and quaternary protein structure by heat/organics (formaline, detergents) Break apart H bonds and disrupt hydrophobic attractions acids/ bases Break H bonds between polar R groups and ionic bonds heavy metal ions React with S-S bonds to form solids: S-Pb-S agitation Stretches chains until bonds break 74 Reversible coagulation Reversible coagulation of proteins – is caused by aqueous solutions of Na+, K+ and NH4+ salts and diluted alcohols, when proteins precipitate out of a solution. After adding water to precipitated proteins, the original protein form is restored (it is called peptization). a protein (a coloid) coagulation a gel peptization 75 25 2016-11-14 Biuret reaction – detection of peptide bond All proteins, peptides with the chain length of at least 3 amino acids give a positive result in this test. This is a typical reaction for identification of peptide bonds. Biuret reaction – detection of peptide bond The Biuret reagent is made of sodium hydroxide (NaOH) and CuSO4 solution. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. Protein purification a processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues 26 2016-11-14 Protein purification based on physico-chemical properties Differences in • • • • • size, shape, and solubility binding affinity isoelectric point charged surface residues and biological activity Protein purification strategies • Size exclusion chromatography • Affinity chromatography (Metal binding, Immunoaffinity chromatography) • Separation based on charge or hydrophobicity • Electrophoresis Size-exclusion chromatography (gel filtration) The column contains a crosslinked polymer with pores of selected size. Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads. The smaller proteins enter the pores and and their path through the column is longer. 81 27 2016-11-14 Affinity chromatography Separates proteins by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand crosslinked to the beads. After nonspecific proteins are washed through the column, the bound protein of particular interest is eluted by a solution containing free ligand. 82 Separation of Proteins Proteins are amphoteric compounds Their net charge therefore is determined by pH of medium in which they are suspended In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards anode in an electrical field Below its isoelectric point, protein is positively charged and migrates towards cathode 83 Electrophoresis of Proteins – Gel electrophoresis allows for the separation of proteins based on charge, size, and shape. – Polyacrylamide gel electrophoresis (PAGE). • Allows for better resolution 84 28 2016-11-14 Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) • Proteins are usually denatured in presence of a detergent such as sodium dodecyl sulfate (SDS) • In denaturing SDS-PAGE separations migration is determined not by intrinsic electrical charge of polypeptide, but by molecular weight 85 Proteins SDS-PAGE Use of sodium dodecyl sulfate (SDS) • Denatures proteins into polypeptide strands • Gives each polypeptide strand an overall negative charge • Proteins studied are strictly being separated by size 86 87 29 2016-11-14 Proteins SDS-PAGE Visualization of proteins in gel • Coomassie Blue • Silver stain – Size of unknown bands can be determined from comparison to protein molecular weight standard 88 30