Proteins biochem

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Proteins biochem

  1. 1. Proteins
  2. 2. Learning Objectives• Understand the importance of the levels of protein structures• Understand the basis for the stability of protein structures• Understand how proteins fold into, and unfold from, their native conformation• Understand the methods employed to analyze proteins
  3. 3. Biologically Active Peptides• Aspartame• Glutathione• Vasopressin• Oxytocin• Enkephalins• Insulin
  4. 4. dipeptide
  5. 5. tripeptide
  6. 6. nanopeptide
  7. 7. nanopeptide
  8. 8. pentapeptide
  9. 9. 2 polypeptides
  10. 10. Protein classification based on: Composition• Simple proteins – no other biomolecules present• Conjugated proteins – presence of metal atom or small organic molecule
  11. 11. Protein classification based on: Solubility• Globular – water soluble; transport function, immune protection and catalysis• Fibrous – water insoluble; structural functions collagen, elastin
  12. 12. Protein classification based on:Function
  13. 13. Protein classification based on:Function
  14. 14. Levels of protein structure• Primary – sequence of amino acids• Secondary – H-bonds bet. backbone C=O and backbone NH (pleated sheet and helix)• Tertiary – interactions of secondary structures• Quaternary – association of polypeptide subunits
  15. 15. Levels of protein structure
  16. 16. Levels of protein structure
  17. 17. Non-polar amino acids
  18. 18. Polar, non-charged amino acids
  19. 19. Negatively-charged amino acids
  20. 20. Positively-charged amino acids
  21. 21. The primary structure reveals the amino acidsequence of each protein/peptide.
  22. 22. Levels of protein structure
  23. 23. Secondary structures• The polar N-H and C=O peptide units in the interior of the protein are held by H-bonds• Two types which are regular structures in protein• a-helix and b-pleated sheet
  24. 24. a-helix features• Coil direction – left handed or right handed• L- amino acids favor the right hand coil• One coil has about 3.6 aa residues; there can be several coils with 650 aa residues(1000Å)• Average length of helix in a globular protein is 15-20Å• H-bonds occur between 1st O of backbone C=O to 13th H atom of backbone NH• The presence of the ff amino acids do not favor the helix formation: Pro, adjacent basic or acidic amino acids, Asn, Tyr, Ser, Thr, Ile and Cys
  25. 25. Knowing the Right Hand from the Left
  26. 26. b-pleated sheet• Two adjacent peptides• Parallel (both NC or CN)• Antiparallel (N to C running in opposite directions)• Antiparallel more common in the structure of proteins• Peptides with this structure are rich in alanine and glycine (silk fiber and spider web)
  27. 27. Supersecondary structures or structural motifs• The clusters are held together by favorable non covalent interactions• Some structural motifs of folded proteins: aa motif; bb motif antiparallel; the Greek key (bbbb) motif; bab motif parallel
  28. 28. Structure of triose phosphate isomerase with several babmotifs combine to form a superbarrel (a) side view (b) topview of the protein
  29. 29. Levels of protein structure
  30. 30. Tertiary structure• Combination of several motifs of secondary structures into a compact arrangement• Noncovalent forces bring about the interactions and stability; – H-bonds, – electrostatic, – hydrophobic, – Van Der Waal’s, – pi-pi complexation between R-side chains – Disulfide bonds occur between Cys residues
  31. 31. Tertiary structures are quite varied
  32. 32. Charged/polar R-groups generally map to surfaces on soluble proteins
  33. 33. Non-polar R-groups tend to be buried in the cores ofsoluble proteins Myoglobin Blue = non-polar R-group Red = Heme
  34. 34. Membrane proteins have adapted to hydrophobic environments
  35. 35. • Water excluded from the hydrophobic interior• Folding of protein occurs after translation in the presence of molecular chaperones• Heat shock proteins (proteins are highly expressed when cells are exposed to increase in temperature) – prevent aggregation of heat-denatured polypeptides• Misfolded proteins aggregate and deposit in certain organs
  36. 36. The diagram shows the role of heat-shock proteins and a chaperonin inprotein folding. As the ribosome moves along themolecule of messenger RNA, a chainof amino acids is built up to form anew protein molecule.The chain is protected againstunwanted interactions with othercytoplasmic molecules by heat-shockproteins and a chaperonin moleculeuntil it has successfully completed itsfolding.
  37. 37. PROTEINDENATURATION
  38. 38. Levels of protein structure
  39. 39. Quaternary structure of proteins• Oligomeric –two or more polypeptide chains; subunits• Homotypic – almost identical subunits• Heterotypic – different subunits• Defines the arrangement and position of each subunit in an intact protein
  40. 40. Examples of other quaternary structures Tetramer Hexamer Filament SSB DNA helicase RecombinaseAllows coordinated Allows coordinated DNA binding Allows complete DNA binding and ATP hydrolysis coverage of an extended molecule
  41. 41. How do biochemists determine the sequence of amino acids?• Sanger technique• Edmann technique• Dansyl chloride technique
  42. 42. Sanger Technique
  43. 43. Edmann Technique
  44. 44. Large Proteins should be sequenced in smaller fragments
  45. 45. Protein isolation• Ion exchange chrom.–based on charge• Gel filtration chrom- based on molecular size• Affinity chrom- selective binding to a specific molecule• Gel electrophoresis- Based on charge and molecular size
  46. 46. ColumnChromatography
  47. 47. Ion-exchangeChromatography
  48. 48. Gel/ Size - exclusionChromatography
  49. 49. AffinityChromatography
  50. 50. Gel Electrophoresis- generally usedsupport medium is cellulose or thin gels made up of either polyacrylamide or agarose.Polyacrylamide is used assupport medium for low molecular weight biochemicals such as amino acid andcarbohydrates whereas agarose for large molecules like proteins Components of the mixture have a uniform charge, electrophoretic mobility depends primarily on size
  51. 51. End of lecture 

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