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Chapter 3 - Amino Acids and Primary Structure of Proteins
Chapter 3 - Amino Acids and Primary Structure of Proteins
Functions of proteins:
1- catalysts - enzymes for metabolic pathways
2- storage and transport - e.g. myoglobin and hemoglobin
3- structural - e.g. actin, myosin
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- specialized functions - e.g. antibodies
There are 20 common amino acids called α -amino acids because they all have an amino
(NH3+) group and a carboxyl group (COOH) attached to C-2 carbon (α 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 α 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.
Amino acids are grouped based upon the properties and structures of side chains.
3) sulfur-containing R groups
methionine - sulfur is internal (hydrophobic)
cysteine - sulfur is terminal --> highly reactive; can form disulfide bonds
5) basic R groups
histidine - hydrophilic side chains - + charged at neutral pH
lysine - “
arginine - strong base
Amide groups can form H bonds with atoms of other polar amino acids.
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All amino acids are have a neutral net charge at physiological pH (7.4).
The α carboxyl and α amino groups and any other ionizable groups determine charge.
Each amino acid has 2 or 3 pKa values (7 amino acids have side chains that are ionizable)
(see Table 3.2). This complicates the basic titration curve, so that there are 3 inflection
points rather than 2.
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.
39810:1 meaning the anion predominates greatly (almost all COOH groups are
ionized).
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At pI of 6.15, there is no net charge (all of the carboxyl groups are unprotonated, and none
of the amino group is unprotonated).
pI = (pKa1 + pKa2)/2
For R groups that are ionizable, the pI is not simply the average!!!
Peptide Bonds
The primary structure of a protein is the linear sequence of amino acids that are covalently
bonded to form a polypeptide chain.
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.
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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.
4- electrophoresis
Separates based upon size and charge (buffer is slightly basic, so most
proteins have negative charge).
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Gives relative quantities of each amino acid, but nothing about order.
First must boil protein in 6N HCl for 24 hours to break peptide bonds.
Problems:
1- acid hydrolysis converts asparagine to aspartic acid and glutamine to glutamic acid
2- also lose some serine, threonine, and tyrosine.
Then take remaining peptide, remove next to last amino acid, etc.
If protein is greater than 50 amino acid residues, must use proteases or chemical reagents
to cleave some of the peptide bonds.
1- cyanogen bromide - reacts with methionine residues - cuts on COOH side
2- proteases
trypsin - cleaves to carboxyl side of lysine and arginine
chymotrypsin - cleaves at aromatic and bulks nonpolar side chains
(phenylalanine, tyrosine, tryptophan)
Staphylococcus aureus V8 protease - cleaves to carboxyl side of
glutamate and aspartate
Need to use at least 2 different cleavage techniques to obtain overlapping sequences using
Edman degradation.