Chapter 2 - Amino Acids and Primary Structure of Proteins Functions of proteins: ( 8 different functions):- (Or classes of proteins according to functions:-) 1- catalysts - enzymes for metabolic pathways 2- storage - albomin as in milk 3- structural - e.g. actin, myosin in muscles 4- mechanical work - movement of flagella and cilia, microtubule movement during mitosis, muscle contraction 5- decoding information - translation and gene expression 6- hormones and hormone receptors 7- immunoproteins - e.g. antibodies 8- and transport - e.g. myoglobin and hemoglobin

Amino acid General Structure Carboxylic acid group Amino group Building blocks of proteins Carboxylic acid group Amino group Side group R gives unique characteristics R side chain I H2H—C —COOH H

Structure of amino acids There are 20 common amino acids called a-amino acids because they all have an amino (NH3+) group and a carboxyl group (COOH) attached to C-2 carbon (a carbon). At pH of 7, amino group is protonated (-NH3+) and carboxyl group is ionized (COO-). The amino acid is called a zwitterion. pKa of a carboxyl group = 1.8 - 2.5 pKa of a amino group = 8.7 - 10.7 The a carbon is chiral or asymmetric ( 4 different groups are attached to the carbon; exception is glycine.) Amino acids exist as stereoisomers (same molecular formula, but differ in arrangement of groups). Designated D(right) or L(left). Amino acids used in nature are of L configuration. carboxylate group at top --> points away sidechain at bottom a amino group orientation determines NH3+ on left = L NH3+ on right = D

Examples of Amino Acids H I H2N—C —COOH H glycine CH3 H alanine

Types of Amino Acids Nonpolar R = H, CH3, alkyl groups, aromatic O Polar ll R = –CH2OH, –CH2SH, –CH2C–NH2, (polar groups with –O-, -SH, -N-) Polar/Acidic R = –CH2COOH, or -COOH Polar/ Basic R = –CH2CH2NH2

Structures of 20 common amino acids: Amino acids are grouped based upon the properties and structures of side chains. 1) aliphatic (R groups consist of carbons and hydrogens) glycine - R=H smallest a.a. with no chiral center alanine - R=CH3 methyl group valine- R = branched; hydrophobic; important in protein folding leucine- R= 4 carbon branched side chain isoleucine- R = 2 chiral centers proline- R = ring; puts bends or kinks in proteins; contains a secondary amino group 2) aromatic (R groups have phenyl ring) phenylalanine - very hydrophobic tyrosine - hydrophobic, but not as much because of polar groups tryptophan - “ Absorb UV light at 280 nm --> used to estimate [protein]

continuation 3) sulfur-containing R groups methionine - sulfur is internal (hydrophobic) cysteine - sulfur is terminal --> highly reactive; can form disulfide bonds 4) side chains with alcohols serine - b-hydroxyl groups --> hydrophilic threonine - “ 5) basic R groups histidine - hydrophilic side chains - + charged at neutral pH lysine - “ arginine - strong base 6) acidic R groups and amide derivatives aspartate - b carboxyl group - confer - charges on proteins glutamate - g carboxyl group asparagine - amide of aspartate - side groups uncharged --> polar glutamine - amide of glutamate - “ Amide groups can form H bonds with atoms of other polar amino acids.

Ionization of Amino Acids All amino acids are have a neutral net charge at physiological pH (7.4). The a carboxyl and a amino groups and any other ionizable groups determine charge. At a given pH, amino acids have different net charges. Can use titration curves for amino acids to show ionizable groups. The isoelectric point (pI) is the pH at which the amino acid has no net charge = zwitterion. If pH > pI, amino acid would be negatively charged. If pH < pI, amino acid would be positively charged. If pH = pI, amino acid would have no charge.

pH and ionization Positive ion zwitterion Negative ion Low pH neutral pH High pH

Amino Acids as Acids and Bases Ionization of the –NH2 and the –COOH group Zwitterion has both a + and – charge Zwitterion is neutral overall + NH2–CH2–COOH H3N–CH2–COO– glycine Zwitterion of glycine

Essential Amino Acids 10 amino acids not synthesized by the body arg, his, ile, leu, lys, met, phe, thr, trp, val Must obtain from the diet All in diary products 1 or more missing in grains and vegetables

Peptide Bonds The primary structure of a protein is the linear sequence of amino acids that are covalently bonded to form a polypeptide chain. Formed by condensation reaction in which a molecule of water is removed. Each amino acid residue is called by replacing -ine or -ate with -yl glycine ---> glycyl The peptide bond is a planar bond with no rotation around C-N axis. If is also in the trans form. Will talk about the consequences later.

The Peptide Bond Amide bond formed by the –COOH of an amino acid and the –NH2 of the next amino acid O CH3 + | | + | NH3–CH2–COH + H3N–CH–COO– O CH3 + | | | NH3–CH2–C – N–CH–COO– | peptide bond H

Peptides Amino acids linked by amide (peptide) bonds Gly --- Lys --- Phe --- Arg --- Ser H2N- -COOH end Peptide bonds end Glycyllysylphenylalanylarginylserine

Protein Purification Techniques All work done at 4oC to minimize degradation. 1- preparation of the protein solution With appropriate buffer, must first disrupt cells by mechanical homogenization with either detergent and/or enzyme treatment. Use a centrifuge to separate into pellet and supernatant. 2- fractionation (relies on protein solubility differences) Use ammonium sulfate (salt) --> interferes with noncovalent bonds between protein and other molecules. Remember solubility is based upon interactions of molecule with water molecules via hydrogen bonds. Different proteins precipitate out at different [salt]. Use centrifuge to remove precipitated protein --> resuspend in buffer. Use dialysis to change out solvent and get rid of NH4SO4.

Continuation 3- chromatography Further fractionates proteins based upon protein’s interaction with matrix. Most commonly used is column chromatography. Uses beads or cellulose fibers. Protein solution is washed through column. Eluate collected and assayed for protein. There are three types of column chromatography: 1) ion-exchange (anion or cation) Separates based upon protein charge. Elute by changing [salt]. 2) gel filtration Uses porous resin. Separates based upon protein size. 3) affinity chromatography Attach ligand to matrix. Can substrate, antibody, etc. Eluate using high [ligand] or high [salt]. Results in 1000-10,000 fold purification.

Continuation 4- electrophoresis Separates proteins based upon migration in an electric field. PAGE - polyacrylamide gel electrophoresis Uses acrylamide as gel matrix. Separates based upon size and charge (buffer is slightly basic, so most proteins have negative charge). SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis Uses SDS and 2-mercaptoethanol. Separation based upon size only. For both, must stain gel to visualize proteins. Bands can be cut out of gel, protein electroeluted, and purified.