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Proteins Proteins Presentation Transcript

  • PROTEINS M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.
  • Amino Acids, Peptides, and Proteins 1 . 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 View slide
  • Some Functions of Proteins 1 . Light : the result of reaction involving the protein luciferin and ATP, catalyzed by the enzyme luciferase. View slide
  • 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 absorption 2. Wave length; A280 3. 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 (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