Proteins

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Transcript of Proteins

  • 1.PROTEINS M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.

2. 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. 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 4. Some Functions of Proteins 1 . Light : the result of reaction involving the protein luciferin and ATP, catalyzed by the enzyme luciferase. 5. 2. Oxygen transport function : Red blood cell, hemoglobin 6. 3. Structural Proteins : Hair , horn, wool 7. General Structure of Amino Acid 1 . Amino Acids 8. Lysine : Basic Amino Acid 9. Stereoisomerism in -Amino Acids Enantiomers : Nonsuperimposable mirror image 10. Steric Relationship of The Stereoisomers of Alanine to The Absolute Configuration of L- and D- Glycelaldehyde 11. 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) 12. Disulfide bond formation 1. Bridge formation between proteins 2. Oxidation-reduction reaction 3. Insulin 2 interdisulfide bridges, one intradisulfide bridge 13. Nonstandard amino acids in proteins 14. Amino Acid Can Act as Acid and Base ** Zwitterion . dipolar ion ** Can act as acid (proton donor) and base (proton acceptor) ** Amphoteric (ampholytes) 15. Absorption of light by molecules Spectrophotometer Wave length of light. Ultrviolet 200- 350nm Visible 400-700 Infra red 700- 16. 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 17. 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. 18. Glutamic Acid pI= pK1 + pKR / 2 = 2.19 + 4.25 /2 = 3.22 19. Histidine pI = pK2 + pKR / 2 = 9.17 + 6.0 = 7.59 20. 2 . Peptides and Proteins Oligopeptide :a few amino acids Polypeptide : many amino acids Amino terminal- N-terminal- Carboxyl terminal- C-terminal 21. Pentapeptide Ser-Gly-Tyr-Ala-Leu 22. 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 23. 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 24. 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 25. 1. Column Chromatography 26. (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 27. (b) Size-exclusion Chromatography(Gel Filtration) 1. Protein size 2. Buffer pH, Salt --- No effect 3. Polymer beads---- no charged 28. (c) Affinity Chromatography 1. Binding specificity 2. Ligands 3. Salt concentration 4. Polymer beads---- ligand attached 29. 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 30. 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 31. 1. Bind to proteins by hydrophobic interaction 2. Make proteins as negatively charged mass 3. So, separated on bases of mass (size) 32. (a) Estimation of Molecular Weight of Proteins( SDS Gel Electrophoresis) 33. (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 34. (c) Two Dimensional Electrophoresis Isoelectric focusing SDS gel electrophoresis 35. Two Dimensional Electrophoresis of E. coli Proteins - more than 2,000 proteins were visualized 36. 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: 1mol/min/at 25C Specific Activity: number of enzyme units/mg protein Specific activity increased 37. 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 38. Frederick Sanger 39. Steps in Sequencing a Polypeptide Steps : Determination of amino acid composition Identification of N-terminal residue(Sangers reagent) Entire sequence (Edman degradation) Sangers reagent Edman reagent 40. 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 41. Correspondence of DNA and Amino Acids Proteome : to describe the entire proteins complement encoded by an organisms DNA