Amino Acids: The Building Blocks of Life

Protein are polymers of -amino acidsThe amino acids used to make proteins are2-aminocarboxylic acids.The (alpha) carbon is the carbon to which afunctional group is attached.

Properties of amino acids:structure and chemical functionalitychiralityacid-base propertiescapacity to polymerize

Proteins in the Diet9 of the 20 amino acids must be obtained from thediet. These are referred to as the essential aminoacids. Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Proteins are also the major source of nitrogen in thediet

Properties of amino acids:Aliphatic chains: Gly, Ala, Val, Leu, and Ile HydrophibicityHydroxyl or sulfur side chains: Ser, Thr, Cys, MetAromatic: Phe, Trp, TyrBasic: His, Lys, ArgAcidic and their amides: Asp, Asn, Glu, Gln

Amino acids Classified according to their capacity to interact with water 4 classes: NON POLAR, POLAR, ACIDIC AND BASIC Non polar amino acids contain hydrocarbon R groups R groups do not have (+) or (-) charges and interact poorly with water 2 types of hydrocarbon chains: aliphatic and aromatic

Non-Polar Side Chains: Side chains which have pure hydrocarbon alkyl groups (alkane branches) or aromatic (benzene rings) are non-polar. Examples include valine, alanine, leucine, isoleucine, phenylalanine. The number of alkyl groups also influences the polarity. The more alkyl groups present, the more non-polar the amino acid will be. This effect makes valine more non-polar than alanine; leucine is more non-polar than valine.

Polar Side Chains:Side chains which have various functional groups such asacids, amides, alcohols, and amines will impart a more polarcharacter to the amino acid.The ranking of polarity will depend on the relative ranking ofpolarity for various functional groupsIn addition, the number of carbon-hydrogens in the alkane oraromatic portion of the side chain should be considered alongwith the functional group.

>Example: Aspartic acid is more polar than serine because anacid functional group is more polar than an alcohol group.

>Example: Serine is more polar than tyrosine, since tyrosinehas the hydrocarbon benzene ring.

Acid - Base Properties of Amino Acids: Acidic Side Chains: If the side chain contains an acid functional group, the whole amino acid produces an acidic solution. Normally, an amino acid produces a nearly neutral solution since the acid group and the basic amine group on the root amino acid neutralize each other in the zwitterion. If the amino acid structure contains two acid groups and one amine group, there is a net acid producing effect. The two acidic amino acids are aspartic and glutamic

Basic Side Chains: If the side chain contains an amine functional group, the amino acid produces a basic solution because the extra amine group is not neutralized by the acid group.Amino acids which have basic side chains include:lysine, arginine, and histidine.

Hydrophobic Amino Acids (aliphatic)

Hydrophobic Amino Acids (aromatic) all very hydrophobic Some contain aromatic group Absorb UV at 280 nm

Sulfur Containing Amino AcidsMethionine (Met, M) startamino acid, very hydrophobicCysteine (Cys,C) sulfur inform of sulfhydroyl, importantin disulfide linkages, weak acid,can form hydrogen bonds.

Charged Amino Acids Asp and Glu are acidic amino acids Contain carboxyl groups Negatively charged at physiological pH, present as conjugatebases Hydrophillic nitrogenous bases Carboxyl groups function as Positively charged at nucleophiles in some enzymatic physiological pH reactions Histidine imidazole ring protonated/ionized, only amino acid that functions as buffer in physiol range. Lysine - diamino acid, protonated at pH 7.0 Arginine - guianidinium ion always protonated, most basic amino acid

Polar Amino Acids 1

Polar Amino Acids 2

Classification of Amino Acids by Polarity POLAR Acidic Neutral Basic Asp Asn Ser Arg Tyr Cys His Glu Gln Thr Lys Gly POLAR Ala Ile Phe Trp NON- Val Leu Met Pro Polar or non-polar, it is the bases of the amino acid properties. Juang RH (2003) Biochemistry

Functional significanceHydrophobic amino acids: encountered in the interiorof proteins shielded from direct contact with waterHydrophillic amino acids: generally found on theexterior of proteins as well as in the active centers ofenzymesImidazole group: act as either proton donor oracceptor at physiological pH Reactive centers of enzymesPrimary alcohol and thiol groups: act as nucleophilesduring enzymatic catalysis Disulfide bonds

Stereochemistry Note that the R group means that the -carbon is a chiral center. All natural amino acids are L-amino acids.

L-Form Amino Acid StructureCarboxylic group - COO Amino group + H3 N H H = Glycine R group CH3 = Alanine Juang RH (2004) BCbasics

Mirror Images of Amino Acid Mirror image Same chemical properties Stereo isomers Juang RH (2004) BCbasics

THE ACID-BASE BEHAVIOUR OF AMINO ACIDS Amino acids are zwitterions: An amino acid has both a basic amine group and an acidic carboxylic acid group.

There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group to leave an ion with both a negative charge and a positive charge. This is called a zwitterion.

Adding an alkali to an amino acid solution increase the pH of a solution of an amino acid by adding hydroxide ions, the hydrogen ion is removed from the -NH3+ group The amino acid would be found to travel towardsthe anode (the positive electrode).

Adding an acid to an amino acid solution decrease the pH by adding an acid to a solution of an amino acid, the -COO- part of the zwitterion picks up a hydrogen ion. the amino acid would move towards thecathode (the negative electrode).

Shifting the pH from one extreme to the other Suppose you start with the ion weve just produced under acidic conditions and slowly add alkali to it. That ion contains two acidic hydrogens - the one in the -COOH group and the one in the -NH3+ group. The more acidic of these is the one in the -COOH group, and so that is removed first - and you get back to the zwitterion.

So when you have added just the right amountof alkali, the amino acid no longer has a netpositive or negative charge. That means that it wouldnt move towards either the cathode oranode during electrophoresis. The pH at which this lack of movement duringelectrophoresis happens is known as theisoelectric point of the amino acid. This pHvaries from amino acid to amino acid.

If you go on adding hydroxide ions, you will get the reaction weve already seen, in which a hydrogen ion is removed from the -NH3+ group.

You can, of course, reverse the whole processby adding an acid to the ion weve just finishedup with. That ion contains two basic groups - the -NH 2group and the -COO- group. The -NH2 group isthe stronger base, and so picks up hydrogenions first. That leads you back to the zwitterionagain.

. . . and, of course, you can keep going by then adding a hydrogen ion to the -COO- group.

Proton Is Adsorbed or Desorbed Proton abundant and small, affects the charge of a molecule lone pair High Low electrons pKa H+Amino N H H+ N H H H Low pKa High O H OCarboxylic C C H+ O O Ampholyte contains both positive and negative groups on its molecule Juang RH (2004) BCbasics

Amino acids are zwitterions:

Acidic environment Neutral environment Alkaline environment pK2 ~ 9 NH2 H+ NH2 H+ NH2R-C-H R-C-H R-C-H COOH COO- COO- pK1 ~ 2 5.5 +1 0 -1 Isoelectric point Juang RH (2004) BCbasics

Amino Acids Have Buffering EffectpH 12 pK2 9 NH2 H+ 6 H-C-R Isoelectric point = pI COO- pK1 + pK2 3 2 pK1 0 [OH] Juang RH (2004) BCbasics

Buffer pHEnvironment pH vs Protein Charge 10 9 8 7 Isoelectric point, pI 6 5 4 3 + 0 - - Net Charge of a Protein Juang RH (2004) BCbasics

H first Aspartic acid HOOC-CH2-C-COOH +1 NH3+ Isoelectric point is the average pK1 = 2.1 of the two pKa flanking the zero net-charged formsecond H 2.1 + 3.9 HOOC-CH2-C-COO- 0 2 = 3.0 NH3+ Isoelectric point pK2 = 3.9 H -2 - OOC-CH2-C-COO- -1 pK3 NH3+ third -1 pK2 pK3 = 9.8 0 H pK1 +1 - OOC-CH2-C-COO- -2 [OH] NH2 Juang RH (2004) BCbasics

Peptide bond formation: Polypeptides are linear polymers composed of amino acids linked together by peptide bonds Peptide bonds are amide linkages formed when unshared electron pair of -carboxyl of another amino acid When 2 amino acids reacted with one another, the product is called a dipeptide. Therefore tripeptide contain 3 amino acid residues, tetrapeptide 4 and so forth

Formation of Peptide Bonds by Dehydration Amino acids are connected head to tailNH2 1 COOH NH2 2 COOH Carbodiimide Dehydration -H2O O NH2 1 C N 2 COOH H Juang RH (2004) BCbasics

By convention, amino acid residue with free NH2 group is called N terminal residue and is written to the left Free COOH on C-terminal is written on the right. Peptides are named by using their amino acid sequences beginning from N-terminal residues, E.g:H2N----Tyr----Ala----Cys----Gly----COOHAbove is a tetratpeptide named tyrosylalanylcysteinylglycine

Polypeptide backbone: Polypeptides are polymers composed of amino acids linked together by peptide/amide bonds Order of amino acids in polypeptide is called amino acid sequence Disulphide bridges formed by oxidation of Cys residues are an important structural element in polypeptides and proteins

Peptides: Less complex than larger protein molecules have significant biological activities E.g: Glutathione, Oxytocin, Vasopressin, substance P and bradykinin Peptides are found in almost all organisms, involved in many important biological processes: -protein DNA synthesis -Drug and environment toxin metabolism -amino acid transport -reducing agent (-SH group of cys) protects cells from destructuve effects of oxidation by reacting with substances such as peroxidase

Disulphide bond 2 cysteine - cystine ; 2 R-SH- R-S-S-R (+2H)(Oxidation reaction)- Intracellular conditions are maintained sufficiently reducing to inhibit formation of most disulfide bonds- Extracellular conditions (as well as those found in some organelles) are more oxidizing, favouring disulphide formation- Thus, extracellular proteins containing cysteines often have disulfides, while intracellular (cytosolic) proteins rarely have disulfides.

Detection, identificationand quantificaton of amino acids and proteins Reaction between the thiol group of cysteine and Ellmans reagent Produce nitrothiobenzoate anion and since this product adsorbs light at 410nm it provides a route for quantifying protein concentration. Other reagents for estimating protein concentration are: ninhydrin, fluorescamine, dansyl chloride, nitrophenols and fluorodinitrobenzene (all react with functional groups)

Protein quantitation:Quick and simple way of estimating protein cncentration1. Spectrophotometric method at 280 nm using quartz cuvettes, absorption mainly due to Trp and Tyr2. Biuret reaction3. Bradford method: widely used4. BCA (Bichinchoninic acid)5. Modified lowry assay6. fluorescamine protein assay Note: to understand the principle behind the reaction used to determine protein concentration, also sensitivity of method used (eg: detection limits of protein assay)Extracts containing protein should be treated with care

1. Absorbance at 280 nm:Principle: Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum. Advantage: Quick estimation, protein not consumed, no additional reagent,incubation needed, no protein standard needed

Historically use biuret reaction: solutionofcopper(II) sulphate in alkaline tartarate solution reacts with peptide bonds to form purple complex absorbing light at540 nm

Disadvantage: considerable error due to varying absoprtion characteristics of protein samples2. Bradford method:Principle: The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. Advantage: relatively fast, fairly accurate

Disadvantage: -The dye reagent reacts primarily with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues. Obviously, the assay is less accurate for basic or acidic proteins. -The Bradford assay is rather sensitive to bovine serum albumin, more so than "average" proteins, by about a factor of two.

3. BCAPrinciple: BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. The advantage of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the copper solution to allow a one step procedure. A molybdenum/tungsten blue product is produced as with the Lowry Disadvantage: greater variability among proteins and the assay is less sensitive

4. Modified lowry assayPrinciple: Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue Advantage: fairly accurate Disadvantage: proteins are consumed and proteins with an abnormally high or low percentage of tyrosine, tryptophan, or cysteine residues will give high or low errors, respectively.

5. Fluorescamine protein assay:Principle: Fluorescamine react with amino acids containing primary amines such as lysine and n-terminal amino acid to yield a highly fluorescent product. Fluoresence measure using a standard fluorometer with the excitation wavelenght at 390 nm and emission at 475nm Advantage: sensitive (nano gram range), fast, reaction is instantaneous Disadvantage: reagents hydrolyzed very rapidly therefore rapid mixing is required to produce reproducible results as fluorescamine react with primary amine, primary amine buffer eg: tris and glycine cant be used

Secondary stucture: Secondary structure of polypeptides consists of several repeating structures most common types: -helix and - pleated sheet -helix and -pleated sheet stabilize by H bonds between carbonyl and NH groups (interactions with other amino acids in close proximity) in polypeptide backbone -helix : rigid, rodlike structure that forms when a polypeptide chain twists into right-handed or left-handed helical conformation.

Nonstandard amino acidschemically modified after they have been incorporated into aprotein (termed a posttranslational modification)- -carboxyglutamic acid, a calcium-binding amino acid residuefound in the blood-clotting protein prothrombin (as well as inother proteins that bind calcium as part of their biologicalfunction).- collagen: Significant proportions of the amino acids incollagen are modified forms of proline and lysine: 4-hydroxyproline and 5-hydroxylysine.- Phosphate molecule to the hydroxyl portion of the R groups ofserine, threonine, and tyrosine. This event is known asphosphorylation and is used to regulate the activity of proteinsin the cell. Serine is the most common in proteins, threonine issecond, and tyrosine is third.

- Glycoproteins are widely distributed in nature and providethe spectrum of functions already discussed for unmodifiedproteins. The sugar groups in glycoproteins are attached toamino acids through either oxygen (O-linked sugars) ornitrogen atoms (N-linked sugars) in the amino acid residues.- Selenocysteine: Although it is part of only a few knownproteins, there is a sound scientific reason to consider this the21st amino acid because it is in fact introduced during proteinbiosynthesis rather than created by a posttranslationalmodification. Selenocysteine is actually derived from theamino acid serine (in a very complicated fashion), and itcontains selenium instead of the sulfur of cysteine.

A helix has the following features:every 3.6 residues make one turn,the distance between two turns is 0.54 nm,the C=O (or N-H) of one turn is hydrogen bonded to N-H (orC=O) of the neighboring turn.Hydrogen bonds play a role in stabilizing the a helixconformation. However, the size and charges of sidechainsare also important factors. Alanine has a greater propensityto form a helices than proline.

The hydrogen bonds that stabilize the helix are parallel to the long axis of the helix.

Beta strandIn a beta strand, the torsion angle of N-Ca-C-N in thebackbone is about 120 degrees. The following figure showsthe conformation of an ideal b strand. Note that thesidechains of two neighboring residues project in theopposite direction from the backbone

Beta sheetA beta sheet consists of two or more hydrogen bonded bstrands. The two neighboring b strands may be parallel ifthey are aligned in the same direction from one terminus (Nor C) to the other, or anti-parallel if they are aligned in theopposite direction.

Structural motif (supersecondary structure):a structural motif is a three-dimensional structural element or fold within the chain, which appears also in a variety of other molecules. In the context of proteins, the term is sometimes used interchangeably with "structural domain," although a domain need not be a motif nor, if it contains a motif, need not be made up of only one.

Rossman fold: Decarboxylase enzyme

What are domains of proteins? A domain is a basic structural unit of a protein structure-distinct from those that make up the conformation Part of protein that can fold into a stable structureindependently different domains can impart different functions to proteinsProteins can have one too many domains depending onprotein sizeIn an unbranched chain-like biological molecule, such as aprotein or RNA, a structural motif is the three dimensionalstructural element within the chain, which appears also inavariety of other molecules.

Pyruvate kinase

Tertiary structure: 3D conformation as a result of interactions betweeen side chains in their primary structure Hydrophobic intercations: as polypeptide folds, R groups are brought into close proximity Electrostatic interactions: strongest electrostatic interaction between ionic groups of opposite charge H bonds: significant number of H bonds forms within interior of protein, polar amino acids interact with water or with polypeptide backbone Covalent bonds: most important, covalent bonds in tertiary structure are disulfide bridges found in many extracellular proteins

Quaternary structure: Proteins esp high M.W composed of several polypeptide chains Each polypeptide is called a subunit Subunits in a protein complex may be identical or quite different Multisubunit proteins in which some or all subunits are identical are called oligomers Polypeptide units assemble and held together by noncovalent interactions such as -hydrophobic interactions -electrostatic interactions -H bonds -covalent cross links

Hydrophobic interactions play an important role in protein folding as well as covalent crosslinks help stabilize multisubunit proteinsEg: disulfide bridges in immunoglobulins, the desmosine and lysinonorleucine linkages in certain connective tissuesEg: desmosine cross links connects 4 polypepide chains in the rubberlike connective tissue called elastinLysinonorleucine: crosslink structure found in elastin and collagenInteractions between subunit are also affected by binding of ligands

In allostery, control of protei fundtion through ligand binding to specific site in protein triggers conformational change that alters its affinity for other ligandsLigand induced corformational changes in such proteins are called allosteric transitions, ligands which trigger them are called effectors or modulatorsLoss of protein structure: Protein sensitive to environmental factors Disruption of native conformation is called denaturation Factors: physical and chemicalDenaturing agents:1. Strong acids or base2. Organic solvents

Hydrogen bonding in a protein

3. Detergents4. Reducing agents5. Salt concentrations6. Heavy metal ions7. Temperature changes8. Mechanical stress

Antibody family: A family of proteins that can be created to bind almost any molecule Ntibodies (imminoglobulin) are made in response to a foreign molecule i.e: bacteria, virus, pollen..callled and antigen Bind together tightly and therefore inactivates the antigen or marks it for destruction

Protein folding: The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favourable positions Weak non covalent interactions will hold the protein in its functional shape-these are weak and will take many to hold the shape. H bonds form between 1) atoms involved in the peptide bonds 2) peptide bond atoms and R groups, 3) R groupsProtein folding: Protein shape is determined by the sequence of the amino acids The final shape is called the conformation and has the lowest free energy possible

3 main classes of protein folding accessory proteins:Allow protein to fold within few minutes in cell (in vivo)a) Protein disulfide isomerasesb) Peptidyl prolyl ci-trans isomerasesc) Molecular chaperones Denatured proteins may renature or refold if chemical compound that causes denaturation can be removed Molecular chaperons are small proteins that help guide the folding and help keep the new protein from associating with the wrong partner

Useful protein: There are many diferent combinations of amino acids that can make up proteins and that would increase if each one had multiple shape Proteins usually have one useful conformation because otherwise it would not be efficient use of energy available to the system Natural selection has elimited proteins that donot perform a specific function in the cell Have similarities in amino acid sequence and 3d structure Have similar functions such as breakdown proteins but do it differently

Proteins multiple peptides non covalent bonds can form interactions betweenindividual polypeptide chains binding site-where proteins interact with one anotherSubunit-each polypeptide chain of large protein dimer protein made of 2 subunits

Oxygen binding protein: Hemoglobin: Carry O2 in blood from lungs to other tisues in body; function is to supply O2 to cells for oxidative phosphorylation Myoglobin stores O2 in tissues of body, available when cells reuire it; highest concentration of myoglobin in skeletal and cardiac muscle which require large amounts of energyMyoglobin: small protein, 17.8 Kda, made up of 153 amino acids in a single polypeptide

Globular protein have a highly folded compact structure with most of the hydrophobic residues found in the interior while polar residues on surfaces Structure of hemoglobin determined by Max Perutz was the first protein structure determined via x-ray crystallography Secondary structure: -helix, 8 -helices, heme prosthetic group is found in hydrophobic crevice formed by folding of polypeptide chains Hemoglobin made up of 4 polypeptide chains Each have similar 3D of single polypeptide chain in myoglobin even though aino acid sequences differ at 83 % of residues This highlight relatively common theme in protein structure: different primary sequence can specify very similar 3D structures

Major tyoe of hemoglobin found in adults (HbA): Made of 2 diferent polypeptide chains: - -chain: 141 amino acid --chain: 146 amino acid Each chain has 8 -helices, each containing heme prosthetic group; therfore hemoglobin can bind 4 molecules of O2 4 polypeptide chains are 22, consists of 2 and 2 packed tightly together ina tetrahedral array to form spherical shaped molecule held together by multiple noncovalent interactions

Important fibrous proteins: Intermediate filaments of the cytoskeleton -structural scaffold inside the cells -keratin in hair, horns and nails Extracellular matrix -binds cells together to make tissues -secreted from cells and assemble in long fibers -collagen: fiber with a glycine every third amino acid in the protein -Elastin: unstructured fibers that give tissues an elastic characteristic

Fibrous proteins: Typically contain high proportion of regular secondary structures such as -helices and -pleated sheets E.g: alpha-keratin, collagen, silk fibroin alpha-keratin:bundles of helical polypeptides twisted together into large bundles Alpha-keratin found in hair wool, skins, horns, fingernails are alpha-helical polypeptides.

Globular protein:Stabilization of cross linkages Cross linkages can be between 2 parts of a protein or between 2 subunits Disulphide bonds (-S-S-) form between adjacent SH groups on the amino acid cystein

Proteins at work: Conformation of a protein gives it a unigque function To work proteins must interact with other molecules, usually 1 or a few molecules from the thousands . Ligand: the molecule that a protein can bind Binding site -part of a protein that interacts with the ligand -consists of a cavity formed by a specific arrangment of amino acids The binding site forms when amino acids from within the protein come together The remaining sequence may play a role in regulating the proteins activity

Chemical characteristic of proteins: Proteins have ionic and hydrophobic sites both internally (within folds of tertiary structure) and on surfaces where primary structures come in contact with the environment Ionic sites are provided by charged amino acids at physiological pH and by covalentl attached modifying group (eg: carbohydrates and phosphate) Net charge on protein contributed by free alpha-amino of N- terminal residue, free alpha-carbonyl group of c terminal residue, ionizable R groups and unique array of modifications attached to proteins At isoelectric point (pI): no of (+) and (-) charges on protein are equal. Protein is electrically neutral Protein has net (+) at pH values below its pI and (-) charge above is pI

Blue: positive charge, red: negative charge