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 pathways2- storage - albomin as in milk3- structural - e.g. actin, myosin in muscles4- mechanical work - movement of flagella and

cilia, microtubule movement during mitosis, muscle contraction

5- decoding information - translation and gene expression

6- hormones and hormone receptors7- immunoproteins - e.g. antibodies

8- and transport - e.g. myoglobin and hemoglobin

Amino acid

General Structure• Building blocks of proteins• Carboxylic acid group• Amino group• Side group R gives unique characteristics

R side chain I

H2H—C —COOH I

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 I

H glycine

CH3

I

H2N—C —COOH I

H alanine

Types of Amino Acids

Nonpolar R = H, CH3, alkyl groups, aromatic

OPolar 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 centeralanine - R=CH3 methyl groupvaline- R = branched; hydrophobic; important in protein foldingleucine- R= 4 carbon branched side chainisoleucine- R = 2 chiral centersproline- R = ring; puts bends or kinks in proteins; contains a secondary amino group

2) aromatic (R groups have phenyl ring)phenylalanine - very hydrophobictyrosine - hydrophobic, but not as much because of polar groupstryptophan - “Absorb UV light at 280 nm --> used to estimate [protein]

continuation3) sulfur-containing R groups

methionine - sulfur is internal (hydrophobic)cysteine - sulfur is terminal --> highly reactive; can form disulfide bonds

4) side chains with alcoholsserine - b-hydroxyl groups --> hydrophilicthreonine - “

5) basic R groupshistidine - hydrophilic side chains - + charged at neutral pHlysine - “arginine - strong base

6) acidic R groups and amide derivativesaspartate - b carboxyl group - confer - charges on proteinsglutamate - g carboxyl group asparagine - amide of aspartate - side groups uncharged --> polarglutamine - 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 solutionWith 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- chromatographyFurther 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 filtrationUses porous resin.Separates based upon protein size.

3) affinity chromatographyAttach 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.