Amino Acids and Proteins 2024 PDF

Amino acids and Proteins Kotelawala Defence University 2024  General structure of an amino acid The central carbon atom is designated as the  - carbon Four groups are attached to it - A basic ami...

Amino acids and Proteins Kotelawala Defence University 2024  General structure of an amino acid The central carbon atom is designated as the  - carbon Four groups are attached to it - A basic amino group (-NH2) - An acidic carboxyl group (-COOH) - A hydrogen atom - A side chain (-R) R – group gives each amino acid its identity α-carbon NH2 group COOH group Side chain (R Group)  At physiologic pH (~ pH = 7.4) The carboxyl group is dissociated forming the negatively charged carboxylate ion (COO-) The amino group is protonated + (NH3 ) Side groups determine properties of amino acids Amino acids are classified as Nonpolar (hydrophobic) with hydrocarbon side chains. Polar uncharged (hydrophilic) with polar or ionic side chains. Acidic (hydrophilic) with acidic side chains. Basic (hydrophilic) with NH2 side chains  Nonpolar Polar Acidic Basic Amino acids with non polar side chains A non-polar amino acid has an R group that is H, an alkyl group, or aromatic. Copyright © 2007 by Pearson Education, Inc. Publishing as Benjamin Cummings Amino acids with non polar side chains Side chains do not gain or lose protons Side chains do not participate in ionic or hydrogen bonding Side chains DO involve in hydrophobic interactions Tend to cluster together in the interior of the protein when it is in an aqueous (polar) environment Hydrophobic & van der waals interactions  Amino acids with polar side chains A polar amino acid has an R group that is an alcohol, thiol, or amide. Neutral at physiological pH Participate in H bonding OH groups act as a site of attachment of phosphates, oligosachcharides Cys-SH plays an important in enzyme active sites and forms disulfide bonds Copyright © 2007 by Pearson Education, Inc. Publishing as Benjamin Cummings  Amino acids with acidic side chains R groups have –COO− R groups are negatively charged at physiological pH Proton donors at physiological pH Aspartic acid Glutamic acid  Amino acids with basic side chains Histidine Lysine Arginine R groups have - NH3+ R groups are positively charged at neutral pH Amino acids are highly hydrophilic  Proline Has a ring structure and a secondary amine group CH2 CHCOO - CH2 NH2 + CH2  L and D amino acids Asymmetric carbon atom (Except glycine) Two isomers (non super imposable mirror images) –enantiomers Can rotate the plane of polarized light either to the right (dextrorotatory) or to the left (levorotatory) Only L – amino acids are present in human proteins Additional amino acids present in proteins Found in cells but do not take part in protein synthesis In free state in cells or as derivatives They perform some essential functions in the cell Eg: Homocysteine- Intermediate in methionine biosynthesis Ornithine - Intermediate in biosynthesis of urea  Cystine Disulfide bond formation between two cysteine residues Post translational modifications of amino acids After protein synthesis (translation) Proline hydroxyproline Lysine hydroxylysine Cysteine cystine  Nutritionally essential amino acids Cannot be synthesized at all, or synthesized inadequately to meet body’s needs Must be supplied in the diet Valine, leucine, isoleucine, lysine, methionine, phenylalanine, histidine, tryptophan & threonine  Properties of amino acids Physical properties Soluble in polar solvents such as water & ethanol but insoluble in non-polar solvents Aromatic amino acids (phenylalanine, tryptophan & tyrosine) absorb UV light at wavelength of 280 nm ----- Protein quantification  Acid and base properties lone pair electrons H + Amino N H H + N H H H O H O Carboxylic C C H + O O Ampholyte - contains both positive and negative groups on its molecule Charge on amino acid at different pHs Acidic environment Neutral environment Alkaline environment pK2 ~ 9 NH2 H + NH2 H + NH2 R-C-H R-C-H R-C-H COOH COO - COO - pK1 ~ 2 5.5 +1 0 -1 Isoelectric point The net charge of an amino acid depends upon the pH of the surrounding solution Isoelectric pH (pI) of an amino acid The pH at which an amino acid bears zero net charge At pI it does not move in an electrical field Exists in its zwitter ionic structure CH3 H3N+ -C – COO- Alanine H Isoelectric pH is the pH midway between pKa values on either side of the isoelectric species  Amino acids -COOH -NH2 -R pH two pKa Gly G 2.34 9.60 pK2 Ala A 2.34 9.69 Val V 2.32 9.62 Leu L 2.36 9.68 pKa of Amino Acids pI Ile I 2.36 9.68 Ser S 2.21 9.15 pK1 Thr T 2.63 10.4 pK1 + pK2 Met M 2.28 9.21 2 Phe F 1.83 9.13 Trp W 2.38 9.39 three pKa Asn N 2.02 8.80 pK3 Gln Q 2.17 9.13 Pro P 1.99 10.6 ? Asp D 2.09 9.82 3.86 pK2 Glu E 2.19 9.67 4.25 His H 1.82 9.17 6.0 ? Cys C 1.71 10.8 8.33 pK1 pI ? Tyr Y 2.20 9.11 10.07 Lys K 2.18 8.95 10.53 Arg R 2.17 9.04 12.48 [OH ] - Calculation of isoelectric pH of alanine pH < 2.35 2.35 10 amino acid residues Protein - a large number of amino acid residues  Peptide bond is rigid and planar C H C N O C Partial double bond character Uncharged but polar  Resonant conjugation O O － C C N＋ N H H O H R2 0.124 1 C 0.1 C 0. 1 5 32 6 C-N: 0.149nm.14 C N 0 C＝N: 0.127nm H R1 H  Levels of organization of proteins Primary Assembly PROCESS STRUCTURE Secondary Folding Tertiary Packing Quaternary Interaction  Primary structure The linear sequence of amino acids comprising a protein: AGVGTVPMTAYGNDIQYYGQV Ordered By convention, written from amino end to carboxyl end Unique to each polypeptide Determines which higher structure it assumes Change in one amino acid may lead to mutation in the protein---- Disease---- Eg; sickle cell anemia  The secondary structure The spatial arrangement of amino acids that are near one another Peptide bond------partial double bond character--------Only a few types of secondary structures!! Formed and stabilized by hydrogen bonding, electrostatic and van der Waals interactions  - helix,  - pleated sheet, -bends   - helix The chain is coiled around an imaginary axis It is stabilized by H bonds formed between the H of a th peptide N and carbonyl oxygen of the 4 amino acid residue ahead 3.6 AAs per turn   - helix Side chains of AA residues protrude outward from the helical backbone. The hydrogen-bonds are parallel to the helical axis. Right handed  - helix is found in proteins Naturally occurring amino acids belong to the L – series In natural systems the  - helix is coiled in a right handed manner Proline breaks the  - helical conformation (a) The pyrrolidine side chain group of proline sterically interacts with the side chain group from the amino acid preceding it in the polypeptide sequence (b) proline is unable to form H – bonds.   - pleated sheet Hydrogen bonds are formed between the – NH & - C = O groups of adjacent sheets H-bonds are perpendicular to the backbone  Disordered regions Regions of proteins are not identifiably organized as helixes or pleated sheets are said to be present in random coil conformation. Disordered regions give flexibility to the structure.  Other secondary structures Greek key β-α-β β-hairpin  Tertiary structure Folding of the secondary structure due to interaction between different regions of the polypeptide chain The three - dimensional arrangement of the molecule in space Protein folding : spontaneously or by chaperon proteins  Tertiary Structure The interactions of the R groups give a protein its specific three- dimensional tertiary structure. Copyright © 2007 by Pearson Education, Inc Publishing as Benjamin Cummings  Types of bonds in tertiary structure Hydrophobic bonds: amino acids with hydrophobic R - groups Hydrogen bonds: between amino acid side chains - + Ionic bonding: Side chains of opposite charges (Coo & NH3 ) Covalent bonds: The most common type is the disulphide bond in cystine Haemoglobin subunit Triose phosphate isomerase Immunoglobulin VL domain Tertiary Structure  Quaternary structure The association of separate polypeptide chains oligomers (protomers, monomers or subuniunits) Two or more tertiary units Stabilized by the same interactions found in tertiary structures Eg: Haemoglobin – Has two alpha subunits and two beta subunits. The haeme group in each subunit binds with oxygen for transport in the blood Summary of Protein Structure Summary of Protein Structures Organization of proteins - Domains Domain A fundamental functional unit of peptides e.g. binding, cleaving, spanning sites  Organization of proteins - Insulin Insulin is a hormone secreted by the  - cells of the islets of Langerhan in the pancreas Human insulin 51 amino acids 2 chains linked by disulfide bonds 5800 Dalton molecular weight The structure of insulin  The structure of insulin The three-dimensional structure of insulin is stabilized by disulphide bridges There are 6 cysteines - 3 disulphide bridges are formed between the A and B chain  Organization of proteins - Myoglobin Myoglobin is a globular protein Found in the muscle tissue The primary structure - 153 amino acid residues The secondary structure - eight right handed helixes & short non helical regions at ends The tertiary structure –globular form  Protein folding Determined by the sequence Number of possible conformations Out of them, ONE biologically active, thermodynamcally favorable conformation----Correct fold! Folding occurs very fast Folding begins during translation Either spontaneously or with the help of chaperones  Chaperones Assist proteins to fold properly Act as catalysts to increase the rate of folding Protect exposed regions of proteins from unproductive interactions  Protein mis-folding Improperly folded proteins are degraded Protein mis-folding makes them aggregate e.g. Alzheimer disease Proteins are diverse  Diversity of proteins There are 20 different amino acids to choose from Each amino acid may appear more than once There are many ways of arranging a given number of amino acids Different combinations-----Different numbers-------Different proteins!!!!  Simple and complex proteins Simple proteins - Contain only amino acids Complex proteins - Contain additional non amino acid materials such as haeme, vitamin derivatives, lipid or carbohydrates (a) Chromoproteins (b) Glycoproteins (c) Phosphoprotein (d) Lipoprotein (e) Nucleoproteins  Chromoproteins Proteins + pigmented prosthetic group Hemoglobins, myoglobin, cytochromes and flavoproteins Heme: pigmented prosthetic group - --porphyrin rings + iron  Glycoproteins Protein + carbohydrate Oligosachcharide is attached to the protein By glycosylation reaction  Phosphoproteins Protein + PO4 3- Protein is bonded to a substance containing phosphate/phosphoric acid Milk protein casein  Lipoproteins Protein + lipid Structure contains both proteins and lipids Eg: Lipovitallin of egg yolk, Chylomicrones, HDL, LDL Proteins in the egg yolk..... Egg yolk is rich in lysine and leucine. They are compounds of major egg protein lipovitellin  Nucleoproteins Protein + nucleic acid Protein is structurally associated with nucleic acid Eg: Chromosomes, ribosome Protein RNA 30S Subunit from Thermus thermophilus  Proteins classification According to  Shape  Solubility  Function  Proteins classification: shape Based on their axial ratios (ratios of length to breadth) Globular proteins Fibrous proteins  Axial ratios less than 10 Axial ratios greater than 10  Compactly folded and coiled Polypeptide chains coiled in a spiral or helix and cross  E.g. insulin, plasma linked covalently by albumins, globulins and hydrogen bonds many enzymes Eg. Keratin, myosin, collagen,  Relatively water soluble fibrin Insoluble in water Globular Vs. Fibrous proteins Proteins classification: function Functional proteins Structural protein Functional proteins  Functional proteins Catalytic role - Enzymes Gene regulation - Histones, nuclear proteins Protection - Fibrin, immunoglobulines Regulatory role - Calmodulin Transport - Albumin – transports bilirubin, fatty acids -Haemoglobin – transports oxygen - Lipoproteins – transport various lipids -Transferrin – transports iron Signal transduction – Hormones (insulin), cytokines Storage - ferritin (storage of iron)  Structural protein Structural role - collagen, elastin, keratins Contractile - Actin, myosin  Keratin Two main types  - keratin - in nails, horns, skin, wool etc. - keratin - in fibrin the structural protein of silk  Keratin Two main types - Alpha - keratin - in nails, horns, skin, wool etc. Beta - keratin - in fibrin the structural protein of silk Alpha- keratin Long, fiber-like shapes Contains cystine or cysteine Very hard because of cystine Stretches on heating and when cooled returns to normal size  Collagen Connective tissues Tendons, skin, bone & blood vessels, cornea of the eye Primary structure Nearly one residue out of three is Gly (~ 33%) Proline content is unusually high Unusual amino acids found: Hydroxyproline & hydroxylysine Stabilization -inter chain H bonds Degradation of collagen results in a release of hydroxyproline and hydroxylysine residues --- Appears in urine  Properties of peptides and proteins Molecular weight Proteins have different molecular weights depending on the number, type & modifications of the amino acids Measured in daltons/ kilodaltons Polyelectrolytes: Charged molecules - charged R groups Proteins interact with other substrates  Enzyme – substrate interactions  Antigen - antibody interactions  Hormone – target molecule interactions Buffering action Plays important role in buffering of blood Proteins are charged – can ionize - can function as buffers  Denaturation of proteins Disruption of secondary, tertiary and quaternary structure of protein Cleavage of non – covalent bonds Unfolding of protein molecules Lose of biological activity Denaturing Agents Heat Shaking High pressure Urea and guanidine Acid and alkali (pH) Organic solvents and detergents  Examples of protein denaturation Alcohol and organic solvents Extreme temperatures 70% Alcohol in sterilization Heating of food make their proteins to denature Your hand sanitizer kills germs by denaturing the proteins in them Denaturing agents interfere Shaking/Agitation with normal bonding of protein structures  Effects of denaturing agents Heat – breaks hydrogen bonds and disrupts hydrophobic interactions Urea and guanidine - Interfere with H – bonding between peptide groups and form H – bonds of their own with these groups Acids and alkalis - break hydrogen bonds between polar R- groups and disrupt ionic bonds Organic solvents and detergents- break hydrogen bonds and disrupt hydrophobic interactions Heavy metal ions - react with disulfide bonds to form solids 70% alcohol is used for sterilization The 100% alcohol denatures the proteins in the membrane instantly..... protects the other proteins from further coagulation. Microorganism may survive 70% alcohol is used for sterilization. Penetrates the cell wall of the organism and denatures all proteins and microorganism dies  Outcomes of protein denatuartion Loss activity Easy attack by digestive enzymes Decreased solubility and therefore precipitation Techniques used to separate amino acids and proteins Chromatography techniques Gel electrophoresis (Isoelectric focusing, SDS PAGE) Western blotting  Principle of chromatography Stationary phase Mobile phase Molecules are partitioned between a stationary phase and a mobile phase Separation depends on the relative interactions between the solute and the stationary phase /mobile phase Affinity Chromatography  Electrophoresis Separation by electrophoresis Charges on proteins allow them to separate in an electric field At given pH, some proteins may be postively charged, some may be negatively charged and some may be neutral Rate of migration depends on the amount of negative charge on the molecules, size and shape An amino acid/protein at its pI does not migrate. SDS-PAGE Protein + Sodium Dodesyl Sulphate Heat at 95 C – 5 min 0 Mix with Gel loading buffer Isoelectric focussing