More Related Content
Amino acid
- 1.
- 2. Introduction • Allpoly/peptides are polymers of amino acids • Naturally, there are more than 300 known amino acids • Only 20 amino acids occur in (mammalian) proteins through protein synthesis. They are also called – basic/primary/standard/proteinogenic – They are almost all L-α-amino acids
- 3. Introduction • Inaddition to the 20 standard amino acids coded by DNA, 2 additional amino acids are also DNA coded in a non-standard manner. – Selenocysteine: in some proteins is coded by the stop codon UGA – Pyrrolysine: used by some methanogenic bacteria in methane producing enzyme systems. Coded for by UAG.
- 4. Introduction • Thestandard amino acids have a wide range of biological functions –Monomers of proteins through the standard genetic code-protein synthesis – Glycine and glutamate are also neurotransmitters. –Tyrosine is involved in the formation of thyroid hormone etc
- 5. Introduction – Standardamino acids also serve as precursors of other molecules • Tryptophan: precursor of the neurotransmitter serotonin • Glycine: is one of the reactants in the synthesis of porphyrins e.g. heme • Arginine: used in the synthesis of (the hormone) nitric oxide
- 6. Introduction • Otheramino acids in proteins are usually formed by post-transcriptional modification of proteins, i.e. after translation. • Such modifications are often important for protein function
- 7. Non std aa:functions and occurrence • Occur as free or in combined states. • Commonly formed as modifications of std aa – E.g taurine is formed by decarboxylation of cysteine – Dopamine is made from tyrosine – Hydroxyproline (in collagen) is made from proline (post transcriptionally)
- 8. Non std aa:functions and occurrence • They serve specialized functions, – e.g. dopamine and γ-amino butyric acid (GABA) are neurotransmitters • They usually occur in the metabolic pathways for standard amino acids, e.g. – Ornithine and citruline: occur in the urea cycle (part of aa breakdown). – Carnitine is used in lipid transport within a cell etc.
- 9. Non std aa:functions and occurrence • Hundreds of other non proteinogenic α- amino acids have been identified in nature –E.g in carbonaceous chondrites (the primitive Murchison meteorite (>79 aa)), –in plants (nisin or alamethicin) etc
- 11. Structure of aminoacids
- 12. Structure of aminoacids • Under normal physiological pH, std aa (except proline), have – an amino group (--NH2) and – a carboxylic acid (--COOH) • Both the amino and the carboxylic acid groups are attached to the same carbon, the tetrahedral α-carbon (refer α-amino acids!).
- 13. Structure of aminoacids • Amino acids are distinguished from one another by their R groups, attached to the α- carbon – One exception is glycine (gly) where the R is hydrogen • The 4th substitution is usually hydrogen • The α-carbon is chiral (except gly), ie is asymmetrical due to having 4 different groups attached (-NH2, -COOH, -R and -H)
- 14. Structure of aminoacids • The side chain R is specific for an amino acid and it confers chemical properties to an aa (weak acids or bases, hydrophiles/-phobes)
- 15. Structure of aminoacids • Understanding the R groups of aa is important in order to devise: – Methods of aa analysis – Methods of aa purification – Characterization and Identification of proteins • e.g. aromatic aa absorb UV light at 275-280nm. Trp has a maximal peak at 280nm etc. proteins can be sequenced and distinguished as in Functional Genomics.
- 16.
- 17. Nomenclature Amino Acid3 letter code 1 letter code Amino Acid 3 letter code 1 letter code Glycine Gly G Threonine Thr T Alanine Ala A Cysteine Cys C Valine Val V Tyrosine Tyr Y Leucine Leu L Asparagine Asn N Isoleucine Ile I Glutamine Gln Q Methionine Met M Aspartic Acid Asp D Proline Pro P Glutamic Acid Glu E Phenyl Phe F Lysine Lys K alanine Tryptophan Trp W Arginine Arg R Serine Ser S Histidine His H
- 18.
- 19. Classification • Rpolarity: 1. hydrophilic (if R is polar) Interact with aqueous environment, Involved in H-bond formation. Found on exterior parts of proteins & reactive sites of enzymes 2. hydrophobic (if R is non-polar) Reside in the interior of proteins,They do not ionizedo not participate in H-bond formation
- 20. Hydrophobic and Neutral at pH=7 Hydrophilic and Neutral at pH=7
- 21. Classification • Acidbase reaction: Depends on ionizability of 1. # of Amino groups 2. # of Carboxylic groups 3. R group properties
- 22. Classification Acidic (TwoCarboxylic Groups) Basic (more than one basic group)
- 23. Amino acid charge At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. -COOH <--------> -COO- + H+ -NH3+ <---------> -NH2 + H+
- 24. Amino acid charge • An amino acid with no ionizable R-group would be electrically neutral at this pH (ie + and – charge balanced). • This species is called a zwitterion.
- 25. Amino acid charge • As in other organic acids, the acidic strength of the carboxyl, amino and ionizable R-groups in amino acids is defined by the pK.
- 26. Amino acid charge • The net charge of an amino acid will thus be the summation of all the charges in the amino acid at a specified pH • Amino acids have different charges at different pH value of the aqueous medium around • When the net charge of an amino acid or protein is zero the pH will be equivalent to the isoelectric point: pI.
- 27. Amino acid charge NOTE: • At its isoelectric pH (pI) an amino acid bears no net charge (The net – and + charge balances)
- 28. Titration curve ofleucine pI = pK1 + pK2 2
- 29. pI = pK2+ pK3 2 Titration of Histidine
- 30. NOTE THAT, YOU • Know the structure of an amino acid. • Know why amino acids are optically active. • Know why some amino acids are called neutral. • Know why some amino acids are called acidic and others basic, hydrophobic/phillic. • Know a zwitterion • Know the definition of an isoelectric point and how this point is calculated for amino acids.
- 31. Peptides and PeptideBonds
- 32. Definitions – Peptidebond=the bond between 2 amino acids – Peptide=a molecule made of the union of aa joined by peptide bonds • Di-ppd= 2 amino acid residues/1 peptide bond • Tri-ppd= 3 amino acid residues/2 peptide bonds • Tetra--=? Etc – Residue=an amino acid in a peptide whose carboxyl group has been used in a peptide bond • Named by replacing the –ate or ine end of aa with –yl • Eg alanine=alanyl, cycteine=cyctenyl etc aspartate= ? Also called aminoacyls
- 33. Peptides IMPORTANCE •Hormones eg insulin • Neurotransmitters/modulators eg • Drugs (antibiotics) eg valinomycin • Beverage sweeterners eg aspartame • Some mediate tumor development
- 34. • Naming ofpeptides • Peptides are named as derivatives of the amino acid with intact –COOH – Eg: Alanyl-aspartyl-histidyl-valine – It means the whole residue sequence is a derivative of valine (valine has an intact –COOH in the above sequence) – Conventinal presentation of peptides: N---------C, – Ie N terminus on the left and the C terminus on the right
- 35. Peptide bond formation • Condensation reaction that result in polymerization of aapeptideprotein • It involves the alfa amino group of one aa and the alfa carboxyl group of the other, with elimination of a water molecule
- 36.
- 37. Electron Resonance Stabilityof peptide bonds • The presence of a carbonyl group in the peptide bond allows for ERS • The peptide bond shows rigidity, ie has a partial double bond character
- 39. Characteristics of thepeptide bond • Due to partial double bond, there is no freedom of rotation around C—N • All 4 atoms of the bond lie on the same plane- it is coplanar – There is free rotation in the rest of the bonds in the peptide backbone – Thus 2/3 of atoms in a peptide are co-planar while 1/3 have free rotation • Peptides are therefore semi-rigid
- 40. Abnormal peptide bonds • Non alfa peptidyl bonds may occur in peptides – Eg in thyrotropin releasing hormone (TRH) where the glutamate is made into a cycle (pyroglutamate) and the carboxyl end is ‘amidated’ (pyrolyl carboxyl) – In glutathione
- 41. Some peptides ofbiological importance • Glutathione – A tripeptide containing glutamic acid, cystein and glycine – Present in RBC and other cells to maintain optimal redox potentials – Anhances/optimizes the action of enzymes (eg glutathione reductase) by preventing oxidation of their –SH to –SS
- 42. Some peptides ofbiological importance • Bradykinin and Kallidin – 9 and 10 residues respectively – From partial hydrolysis of plasma proteins by snake venoms (hemolytic venoms) – Powerful vasodepressors and cardiac inhibitors
- 43. Some peptides ofbiological importance • Tyrocidin and Gramicidin – Cyclic peptides, 10 aa each – They form rings their terminal 2 amino acid residues – They are useful antibiotics
- 44. Some peptides ofbiological importance • Peptide hormones and factors – Posterior Pituitary-oxytocin and vassopressin – Hypothalamic hormones-somatostatin – Intestinal hormones-gastrin – Kidney and brain factors-hypertensin and enkephalins
- 45. • PLEASE NOTE: – Do you know what is a peptide and peptide bond? – Dou you know an amino acid and an amino acid residue? – Dou you know how to name residues and peptides? – Peptide bond formation? – Do you know the features of a peptide bond? – Do you know why peptides are semi-rigid?
- 46. The primary structureof proteins
- 47. Primary structure •Refers to – the number and the order of amino acid residues in the peptide/polypeptide – This is the linear sequence of residues
- 48. Primary structure andbiological • Mutations activity – SNPs: DNA changechange in the order & sometimes the number of amino acid residues. eg 1. Alcohol intolerance in many Asians 2. Susceptibility to malaria (K189M in the ICAM-1 gene susceptibility to cerebral malaria)
- 49. Primary structure Nomenclature • Both 3 letter and single letter systems used to determine primary structures of ppds (remember aa nomenclature) • In the 3 letter system, residues are joined by a dash/line: Gly-Tyr-Ala-Glu-Lys • In the 1 letter system the same ppd becomes: GYAEK, ie no lines
- 50. Primary structure Nomenclature • If uncertain about a residue, it is enclosed in brackets and the possible residues separated by a comma – Thus Gly- (Tyr,Ala)-Glu-Lys
- 51. Charge on peptides • Formation of the peptide bond involves the loss of +1 and -1 =0 charge. • Peptide bonds are neutral at any pH • At pH 7.4, peptides are charged due to – N-terminal group – C-terminal group – Basic or acidic R groups
- 52. Determination of primarystructure • Sanger worked with insulin in the 60s. –Separated the 2 chains, A and B, cleaved the peptides into smaller peptides with overlapping sequences – Using 1-fluoro-2,4-dinitrobenzene, he could remove and identify each aa at a time
- 53. Protein mixture Stagesof sequencing Proteins Peptide mixture Peptides MS analysis MS data Identification separation digestion digestion separation Database search algorithms
- 54. Stages of sequencing • Purify peptides and asses and purify by any suitable technique e.g. SDS-PAGE, chromatography • Homogeneity should be 90-95%
- 56. FAB-MS Schematic Representation Xenon atoms Helium atoms
- 57.
- 58. From public onlinedata banks
- 59. Applications of sequencing To study Protein functions, localization, modifications and protein interactions Also therapeutics and diagnostics
- 60. • DO YOUKNOW – The biological implications of the primary structure of proteins (peptides) – Nomenclature of peptides? – Basic steps of sequencing a peptide? – Importance of peptide sequencing? – The principle of FAB MS in peptide sequencing? • Then GOOD DAY!