PROTEINS

M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar.

Amino Acids, Peptides, and Proteins1 . Amino Acids Share Common Structural Features

1. 20 Amino Acids and Classification

2. Amphoteric Properties and Titration curve

3. Isoelectric Point(pI)

2. Peptides and Proteins 1. Peptide Bond : Oligopeptide, Polypeptide 2. Characteristic Amino Acid Composition 3. Conjugated Proteins 4. Protein Structure : Primary, Secondary, Tertiary Quaternary Structure

3. Working With Proteins

1. Protein Purification : Crude Extract, Fractionation,

Column Chromatography, HPLC, Electrophoresis

4 . Covalent Structure of Proteins 1. Amino Acid Sequencing : Edman Degradation N-terminal, C-terminal determination 2. Breaking disulfide bond, Cleaving polypeptide chain Sequencing of peptide, Ordering peptide fragments Locating disulfide bonds 3. Peptides can be chemically synthesized

Some Functions of Proteins

1 . Light : the result of reaction involving the protein luciferin

and ATP, catalyzed by the enzyme luciferase.

2. Oxygen transport function : Red blood cell, hemoglobin

3. Structural Proteins : Hair , horn, wool

General Structure of Amino Acid

1 . Amino Acids

Lysine : Basic Amino Acid

Stereoisomerism in α-Amino Acids

Enantiomers : Nonsuperimposable mirror image

Steric Relationship of The Stereoisomers of Alanine to The Absolute Configuration of L- and D-Glycelaldehyde

Properties of aromatic amino acids

1. Characteristics of UV absorption2. Wave length; A2803. Phe : phenyl-, Tyr : phenol-, Trp : indole-

** DNA, RNA….. A260 (purine, pyrimidine base)

Disulfide bond formation

1. Bridge formation between proteins

2. Oxidation-reduction reaction

3. Insulin…… 2 interdisulfide bridges, one intradisulfide bridge

Nonstandard amino acids in proteins

Amino Acid Can Act as Acid and Base

** Zwitterion …. dipolar ion** Can act as acid (proton donor) and base (proton acceptor)** Amphoteric (ampholytes)

Absorption of light by molecules

• Spectrophotometer

•Wave length of light…. Ultrviolet 200-350nm

Visible 400-700

Infra red 700-

Titration Curve of Amino Acid

1. First COOH group titrated, then NH3 group

2. Tow buffer zones

3. Amino acid is amphipatic

4. Isoelectric point (pI)

5. Below pI → positive charge,

6. Above pI → negative charge

Effect of the chemical environment on pKa

** The pKa of any functional groups is greatly affected by its chemical environment.

Similar effects can be observed in the active site of enzymes.

Glutamic Acid

pI= pK1 + pKR / 2

= 2.19 + 4.25 /2

= 3.22

Histidine

pI = pK2 + pKR / 2

= 9.17 + 6.0

= 7.59

2 . Peptides and Proteins

Oligopeptide :a few amino acids

Polypeptide : many amino acids

Amino terminal-

N-terminal-

Carboxyl terminal-

C-terminal

Pentapeptide

Ser-Gly-Tyr-Ala-Leu

Tetrapeptide

1. Acid-base behavior of a peptide:

N-terminal, C-terminal, R-groups

2. Peptides have a characteristic titration curve and a characteristic pI value

Levels of structure in proteins

Primary structure of protein : amino acid sequence

Secondary structure of protein : local structure

Tertiary structure of protein : three dimensional structure

Quaternary structure of protein : subunits

Protein Separation and Purification

Why Purification? : to understand the structure and functions of proteins

Purification Procedure : 1. Crude extract 2. Subcellular fractionation

3. Fractionation of proteins---- Size, Charge, pH,

Solubility, Salt concentration, Dialysis

Methods of Protein Purification and Identification:

1. Column Chromatography ---- Ion exchange chromatography

Size-exclusion chromatography

Affinity chromatography

2. Gel Electrophoresis ------- SDS gel electrophoresis

Isoelectric focusing

Two dimensional electrophoresis

(purification)

(Identification)

3. Working with Proteins

1. Column Chromatography

(a) Ion Exchange Chromatography

1. Anion Exchanger--- matrix with cation(+)

Cation Exchanger--- matrix with anion(-)

2. Buffer pH is very important (pI)

3. Salt Effect

(b) Size-exclusion Chromatography(Gel Filtration)

1. Protein size

2. Buffer pH, Salt --- No effect

3. Polymer beads---- no charged

(c) Affinity Chromatography

1. Binding specificity

2. Ligands

3. Salt concentration

4. Polymer beads---- ligand attached

2. Gel Electrophoresis

1. Use electricity

2. Use polyacrylamide gel (polymer)

3. Based on the migration of charged proteins in electric field

4. pI of proteins are very important

5. Charge , mass, and shape of protein are importnat

Visualization of Proteins after Electrophoresis

1. Staining with dye(Coomassie blue, BPB)

2. Destaining with acetic acid solution

3. Smaller and larger charge proteins move faster

1. Bind to proteins by hydrophobic interaction

2. Make proteins as negatively charged mass

3. So, separated on bases of mass (size)

(a) Estimation of Molecular Weight of Proteins

( SDS Gel Electrophoresis)

(b) Isoelectric focusing

1. Determine the pI value of proteins

2. Use ampholyte solution

3. Proteins are distributed along pH gradient according to their pI values

4. pI value of protein---- R-group

(c) Two Dimensional Electrophoresis

Isoelectric focusing SDS gel electrophoresis

Two Dimensional Electrophoresis of E. coli Proteins

- more than 2,000 proteins were visualized

Unseparated Proteins (Enzyme) can be Quantified

Quantitating of Proteins (Enzyme Activity):

1. Overall enzymatic reaction 2. Analytical procedures

3. Cofactors or coenzymes 4. Substrate concentration

5. Optimum pH and temperature

1 Unit of enzyme: 1μmol/min/at 25ºC

Specific Activity:

number of enzyme units/mg protein

Specific activity increased

4. Covalent Structure of Proteins (Primary Structure)

Primary structure→ Amino acid sequence

Different amino acid sequence →different function

Genetic disease →single amino acid change

Similar function protein of different species→

similar sequence of amino acids

Bovine Insulin

Bovine Insulin : 51 amino acid,

3 disulfide bonds

Frederick Sanger

Steps in Sequencing a Polypeptide

Steps : Determination of amino acid composition

Identification of N-terminal residue(Sanger’s reagent)

Entire sequence (Edman degradation)

Sanger’s reagent

Edman reagent

Large Proteins must be Sequenced in Smaller Segments

1. Breaking disulfide bonds

2. Cleaving the Polypeptide Chain

3. Sequencing of Peptides

4. Ordering Peptide Fragments

Correspondence of DNA and Amino Acids

Proteome : to describe the entire proteins complement encoded by an organism’s DNA