PROTEIN

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. 1 . Amino Acids General Structure of Amino Acid

8. Lysine : Basic Amino Acid

9. Stereoisomerism in α-Amino Acids Enantiomers : Nonsuperimposable mirror image

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

16. 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)

17. Disulfide bond formation • Bridge formation between proteins • Oxidation-reduction reaction • Insulin…… 2 interdisulfide bridges, one intradisulfide bridge

18. Nonstandard amino acids in proteins

20. Amino Acid Can Act as Acid and Base ** Zwitterion …. dipolar ion ** Can act as acid (proton donor) and base (proton acceptor) ** Amphoteric (ampholytes)

21. Absorption of light by molecules • Spectrophotometer • Wave length of light…. Ultrviolet 200-350nm • Visible 400-700 • Infra red 700-

22. Titration Curve of Amino Acid • First COOH group titrated, then NH3 group • Tow buffer zones • Amino acid is amphipatic • Isoelectric point (pI) • Below pI → positive charge, • Above pI → negative charge

23. 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.

24. Glutamic Acid pI= pK1 + pKR / 2 = 2.19 + 4.25 /2 = 3.22

25. Histidine pI = pK2 + pKR / 2 = 9.17 + 6.0 = 7.59

26. 2 . Peptides and Proteins Carboxyl terminal- C-terminal Amino terminal- N-terminal- Oligopeptide :a few amino acids Polypeptide : many amino acids

27. Pentapeptide Ser-Gly-Tyr-Ala-Leu

28. Tetrapeptide • Acid-base behavior of a peptide: • N-terminal, C-terminal, R-groups • 2. Peptides have a characteristic titration curve and a characteristic pI value

29. 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

30. 3. Working with Proteins 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)

31. 1. Column Chromatography

32. (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

33. (b) Size-exclusion Chromatography(Gel Filtration) • Protein size • Buffer pH, Salt --- No effect • Polymer beads---- no charged

34. (c) Affinity Chromatography • Binding specificity • Ligands • Salt concentration • Polymer beads---- ligand attached

35. 2. Gel Electrophoresis • Use electricity • Use polyacrylamide gel (polymer) • Based on the migration of charged proteins in electric field • pI of proteins are very important • Charge , mass, and shape of protein are importnat

36. Visualization of Proteins after Electrophoresis • Staining with dye(Coomassie blue, BPB) • Destaining with acetic acid solution • Smaller and larger charge proteins move faster

37. Bind to proteins by hydrophobic interaction • Make proteins as negatively charged mass • So, separated on bases of mass (size)

38. (a) Estimation of Molecular Weight of Proteins ( SDS Gel Electrophoresis)

39. (b) Isoelectric focusing • Determine the pI value of proteins • Use ampholyte solution • Proteins are distributed along pH gradient according to their pI values • pI value of protein---- R-group

41. (c) Two Dimensional Electrophoresis Isoelectric focusing SDS gel electrophoresis

42. Two Dimensional Electrophoresis of E. coli Proteins - more than 2,000 proteins were visualized

43. 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

44. 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 : 51 amino acid, 3 disulfide bonds Bovine Insulin

45. Frederick Sanger

46. 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

47. Large Proteins must be Sequenced in Smaller Segments • Breaking disulfide bonds • Cleaving the Polypeptide Chain • Sequencing of Peptides • Ordering Peptide Fragments

48. Correspondence of DNA and Amino Acids Proteome : to describe the entire proteins complement encoded by an organism’s DNA

49. THANK YOU