Aug 26 2011


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Aug 26 2011

  1. 1. BT-202 Netaji Subhas Institute of Technology, Dwarka, New Delhi. Dr. Amita Pandey Aug 26, 2011
  2. 2. Learning check! <ul><li>Serine has pK values of 2.19 and 9.21. What is its estimated pI? </li></ul><ul><li>2.19 </li></ul><ul><li>5.70 </li></ul><ul><li>7.00 </li></ul><ul><li>9.21 </li></ul><ul><li>By convention amino terminal residue of a peptide chain is written as the right-most residue. </li></ul><ul><li>True </li></ul><ul><li>False </li></ul>
  3. 3. <ul><li>The alpha amino groups of all amino acids have a charge at pH 7.0. </li></ul><ul><li>positive </li></ul><ul><li>Which ionic species is most prevalent at the pI. </li></ul><ul><li>H 2 NCH 2 COOH </li></ul><ul><li>H 3 N + CH 2 COOH </li></ul><ul><li>H 3 N + CH 2 COO - </li></ul><ul><li>H 2 NCH 2 COO - </li></ul><ul><li>In which pH range is glycine fully protonated. </li></ul><ul><li>Above pH 10.0 </li></ul><ul><li>pH 2.35 - pH 9.78 </li></ul><ul><li>pH 2.35 +1/-1 </li></ul><ul><li>Below pH 1.0 </li></ul>
  4. 4. <ul><li>Which side chain is most likely to be negatively charged @ pH 7.0? </li></ul><ul><li>Arginine </li></ul><ul><li>Aspartic acid </li></ul><ul><li>Tyrosine </li></ul><ul><li>Cysteine </li></ul><ul><li>Which amino acid side chain is NOT aromatic? </li></ul><ul><li>Trytophan </li></ul><ul><li>Methionine </li></ul><ul><li>Tyrosine </li></ul><ul><li>Phenylalanine </li></ul>
  5. 5. Structure of proteins Proteins perform diverse functions in the cell.
  6. 6. Primary Structure <ul><li>Function of a protein depends upon its AA sequence. </li></ul><ul><li>-proteins with different functions have different AA sequence. </li></ul><ul><li>-various genetic disorders have defective proteins. </li></ul><ul><li>-functionally related proteins have similar AA sequence. </li></ul>
  7. 7. Polymorphic proteins <ul><li>Same protein with different amino acid composition is called polymorphic protein. </li></ul><ul><li>-20%-30% proteins in humans are polymorphic. Eg ABO blood group. </li></ul><ul><li>-polymorphism also exists among distantly related species. </li></ul>
  8. 8. First protein Sequence <ul><li>Frederick Sanger (1953) </li></ul><ul><li>AA sequence of hormone </li></ul><ul><li>insulin </li></ul>
  9. 9. End Group Analysis <ul><li>Identify number of terminal AAs </li></ul><ul><ul><li>Number of chains/subunits </li></ul></ul><ul><li>Identify specific AA </li></ul><ul><li>Sanger method (FDNB) </li></ul>
  10. 10. End Group Analysis <ul><li>Dansyl chloride </li></ul><ul><li>Reacts with primary amines </li></ul><ul><ul><li>N-terminus </li></ul></ul><ul><li>Yields dansylated polypeptides </li></ul><ul><li>Dansylated polypeptides hydrolyzed to liberate the modified dansyl AA </li></ul><ul><li>Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence) </li></ul><ul><li>Dabsyl chloride </li></ul>
  11. 11. Edman degradation <ul><li>Only single AA from the N-terminus is cleaved </li></ul><ul><li>Entire protein sequence can be deduced </li></ul><ul><li>Identify using NMR, HPLC, etc. </li></ul><ul><li>Sequenator (entire process for proteins < 100 residues) </li></ul>
  12. 12. Sequencing larger proteins <ul><li>Formation of smaller segments to assist with sequencing </li></ul><ul><li>Process: </li></ul><ul><ul><li>Cleave protein into specific fragments </li></ul></ul><ul><ul><ul><li>Chemically or enzymatically </li></ul></ul></ul><ul><ul><ul><li>Break disulfide bonds </li></ul></ul></ul><ul><ul><li>Purify fragments </li></ul></ul><ul><ul><li>Sequence fragments </li></ul></ul><ul><ul><li>Determine order of fragments and disulfide bonds </li></ul></ul>
  13. 13. Breaking disulfide bond <ul><li>performic acid </li></ul><ul><li>Cys residues form </li></ul><ul><li>cysteic acid </li></ul><ul><li>Acid can oxidize </li></ul><ul><li>other residues, so </li></ul><ul><li>not ideal </li></ul>
  14. 14. Breaking disulfide bond <ul><li>β -2-Mercaptoethanol </li></ul><ul><li>(HSCH 2 CH 2 OH) </li></ul><ul><li>Dithiothreitol (DTT) </li></ul><ul><ul><li>(HSCH 2 CH(OH)CH(OH)CH 2 SH) </li></ul></ul>
  15. 15. Enzymatic and Chemical Cleavage <ul><li>Enzymatic </li></ul><ul><ul><li>Endopeptidases </li></ul></ul><ul><li>Chemical </li></ul><ul><ul><ul><li>-Cyanogen bromide (CNBr) </li></ul></ul></ul>
  16. 16. peptide fragments Sequencing Edman procedure Ordering of peptides Cleaved again with different enzyme or chemical
  17. 18. Learning check <ul><li>A protein is cleaved with cyanogen bromide to yield the following sequences: </li></ul><ul><ul><li>Arg-Ala-Tyr-Gly-Asn </li></ul></ul><ul><ul><li>Leu-Phe-Met </li></ul></ul><ul><ul><li>Asp-Met </li></ul></ul><ul><li>The same protein is cleaved with chymotrypsin to yield the following sequences: </li></ul><ul><ul><li>Met-Arg-Ala-Tyr </li></ul></ul><ul><ul><li>Asp-Met-Leu-Phe </li></ul></ul><ul><ul><li>Gly-Asn </li></ul></ul><ul><li>What is the sequence of the protein? </li></ul><ul><li>Asp-Met-Leu-Phe-Met-Arg-Ala-Tyr </li></ul>
  18. 19. Protein sequencing <ul><li>Mass spectrometry </li></ul><ul><li>- 20-30 AA long fragments. </li></ul><ul><li>Determination of protein sequence from DNA sequence </li></ul><ul><li> </li></ul>
  19. 20. Genome sequencing organism Size of genome Year Haemophilus 1.8 Mb 1995 Saccharomyces 12.1 Mb 1996 C. elegans 100Mb 1998 Fruit fly 139 Mb 2005 Human 3.5 Gb 2006
  20. 21. Consensus sequences
  21. 22. Synthesis of peptides Three ways to obtain a peptide are -from the tissue -genetic engineering -chemical synthesis
  22. 23. Chemical synthesis 9-fluorenylmethoxycarbonyl
  23. 25. Proteins <ul><li>Make up about 15% of the cell </li></ul><ul><li>Have many functions in the cell </li></ul><ul><ul><li>Enzymes </li></ul></ul><ul><ul><li>Structural </li></ul></ul><ul><ul><li>Transport </li></ul></ul><ul><ul><li>Motor </li></ul></ul><ul><ul><li>Storage </li></ul></ul><ul><ul><li>Signaling </li></ul></ul><ul><ul><li>Receptors </li></ul></ul><ul><ul><li>Gene regulation </li></ul></ul>
  24. 26. Protein folding <ul><li>The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions </li></ul><ul><li>Weak non-covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape </li></ul>
  25. 27. Non-covalent bonds in protein folding
  26. 28. Globular protein
  27. 29. Hydrogen Bonds in Proteins
  28. 30. Protein folding <ul><li>Proteins shape is determined by the sequence of the amino acids </li></ul><ul><li>The final shape is called the conformation and has the lowest free energy possible </li></ul><ul><li>Denaturation is the process of unfolding the protein </li></ul><ul><ul><li>with heat, pH or chemical compounds </li></ul></ul>
  29. 31. Protein folding <ul><ul><li>Renaturation is the process of protein regaining its native conformation </li></ul></ul><ul><ul><li>Eg. Ribonuclease </li></ul></ul><ul><li>Molecular chaperones are small proteins that help guide the folding and can help keep the new protein from associating with the wrong partner </li></ul>
  30. 32. Protein folding  -helix – protein turns like a spiral – fibrous proteins (hair, nails, horns)  -sheet – protein folds back on itself as in a ribbon –globular protein
  31. 33.  Sheets <ul><li>Core of many proteins is the  sheet </li></ul><ul><li>Form rigid structures with the H-bond </li></ul><ul><li>Can be of 2 types </li></ul><ul><ul><li>Anti-parallel – run in an opposite direction of its neighbor (A) </li></ul></ul><ul><ul><li>Parallel – run in the same direction with longer looping sections between them (B) </li></ul></ul>
  32. 34.  Helix <ul><li>Formed by a H-bond between every 4 th peptide bond – C=O to N-H </li></ul><ul><li>Usually in proteins that span a membrane </li></ul><ul><li>The  helix can either coil to the right or the left </li></ul><ul><li>Can also coil around each other – coiled-coil shape </li></ul>
  33. 35. Protein structure
  34. 36. Domains <ul><li>A domain is a basic structural unit of a protein structure – distinct from those that make up the conformations </li></ul><ul><li>Part of protein that can fold into a stable structure independently </li></ul><ul><li>Different domains can impart different functions to proteins </li></ul><ul><li>Proteins can have one to many domains depending on protein size </li></ul>
  35. 37. Domains
  36. 38. Protein Families <ul><li>Have similarities in amino acid sequence and 3-D structure </li></ul><ul><li>Have similar functions such as breakdown proteins but do it differently </li></ul>
  37. 39. Proteins – Multiple Peptides <ul><li>Non-covalent bonds can form interactions between individual polypeptide chains </li></ul><ul><ul><li>Binding site – where proteins interact with one another </li></ul></ul><ul><ul><li>Subunit – each polypeptide chain of large protein </li></ul></ul><ul><ul><li>Dimer – protein made of 2 subunits </li></ul></ul><ul><ul><ul><li>Can be same subunit or different subunits </li></ul></ul></ul>
  38. 40. Single Subunit Proteins
  39. 41. Different Subunit Proteins <ul><li>Hemoglobin </li></ul><ul><ul><li>2  globin subunits </li></ul></ul><ul><ul><li>2  globin subunits </li></ul></ul>
  40. 42. Protein Assemblies <ul><li>Proteins can form very large assemblies </li></ul><ul><li>Can form long chains if the protein has 2 binding sites – link together as a helix or a ring </li></ul><ul><li>Actin fibers in muscles and cytoskeleton – is made from thousands of actin molecules as a helical fiber </li></ul>
  41. 43. Types of Proteins <ul><li>Globular Proteins – most of what we have dealt with so far </li></ul><ul><ul><li>Compact shape like a ball with irregular surfaces </li></ul></ul><ul><ul><li>Enzymes are globular </li></ul></ul><ul><li>Fibrous Proteins – usually span a long distance in the cell </li></ul><ul><ul><li>3-D structure is usually long and rod shaped </li></ul></ul>
  42. 44. Important Fibrous Proteins <ul><li>Intermediate filaments of the cytoskeleton </li></ul><ul><ul><li>Structural scaffold inside the cell </li></ul></ul><ul><ul><ul><li>Keratin in hair, horns and nails </li></ul></ul></ul><ul><li>Extracellular matrix </li></ul><ul><ul><li>Bind cells together to make tissues </li></ul></ul><ul><ul><li>Secreted from cells and assemble in long fibers </li></ul></ul><ul><ul><ul><li>Collagen – fiber with a glycine every third amino acid in the protein </li></ul></ul></ul><ul><ul><ul><li>Elastin – unstructured fibers that gives tissue an elastic characteristic </li></ul></ul></ul>
  43. 45. Collagen and Elastin
  44. 46. Stabilizing Cross-Links <ul><li>Cross linkages can be between 2 parts of a protein or between 2 subunits </li></ul><ul><li>Disulfide bonds (S-S) form between adjacent -SH groups on the amino acid cysteine </li></ul>
  45. 47. Proteins at Work <ul><li>The conformation of a protein gives it a unique function </li></ul><ul><li>Ligand – the molecule that a protein can bind </li></ul><ul><li>Binding site – part of the protein that interacts with the ligand </li></ul><ul><ul><li>Consists of a cavity formed by a specific arrangement of amino acids </li></ul></ul>
  46. 48. Ligand Binding
  47. 49. Formation of Binding Site <ul><li>The binding site forms when amino acids from within the protein come together in the folding </li></ul><ul><li>The remaining sequences may play a role in regulating the protein ’s activity </li></ul>
  48. 50. Antibody Family <ul><li>A family of proteins that can be created to bind to almost any molecule </li></ul><ul><li>Antibodies (immunoglobulins) are made in response to a foreign molecule ie. bacteria, virus, pollen… called the antigen </li></ul><ul><li>Bind together tightly and therefore inactivates the antigen or marks it for destruction </li></ul>
  49. 51. Antibodies <ul><li>Y-shaped molecules with 2 binding sites at the upper ends of the Y </li></ul><ul><li>The loops of polypeptides on the end of the binding site are what imparts the recognition of the antigen </li></ul><ul><li>Changes in the sequence of the loops make the antibody recognize different antigens - specificity </li></ul>
  50. 52. Antibodies
  51. 53. Binding Strength <ul><li>Can be measured directly </li></ul><ul><li>Antibodies and antigens are mixing around in a solution, eventually they will bump into each other in a way that the antigen sticks to the antibody, eventually they will separate due to the motion in the molecules </li></ul><ul><li>This process continues until the equilibrium is reached – number sticking is constant and number leaving is constant </li></ul><ul><li>This can be determined for any protein and its ligand </li></ul>
  52. 54. Equilibrium Constant <ul><li>Concentration of antigen, antibody and antigen/antibody complex at equilibrium can be measured – equilibrium constant (K) </li></ul><ul><li>Larger the K the tighter the binding or the more non-covalent bonds that hold the 2 together </li></ul>
  53. 55. Enzymes as Catalysts <ul><li>Enzymes are proteins that bind to their ligand as the 1 st step in a process </li></ul><ul><li>An enzyme ’s ligand is called a substrate </li></ul><ul><ul><li>May be 1 or more molecules </li></ul></ul><ul><li>Output of the reaction is called the product </li></ul><ul><li>Enzymes can repeat these steps many times and rapidly, called catalysts </li></ul>
  54. 56. Enzymes at Work <ul><li>Lysozyme is an important enzyme that protects us from bacteria by making holes in the bacterial cell wall and causing it to break </li></ul><ul><li>Lysozyme adds H 2 O to the glycosidic bond in the cell wall </li></ul><ul><li>Lysozyme holds the polysaccharide in a position that allows the H 2 O to break the bond – this is the transition state – state between substrate and product </li></ul><ul><li>Active site is a special binding site in enzymes where the chemical reaction takes place </li></ul>
  55. 57. Lysozyme <ul><li>Non-covalent bonds hold the polysaccharide in the active site until the reaction occurs </li></ul>
  56. 58. Features of Enzyme Catalysis