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Gro els


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Gro els

  1. 1. GroEL-GroES Chaperonin Complex PDB ID = 1AON
  2. 2. What are Chaperonins? <ul><ul><li>Large double-ring-shaped protein complexes </li></ul></ul><ul><ul><li>whose role in vivo is to assist protein folding </li></ul></ul>
  3. 3. Where can chaperonins be found? <ul><li>Classified by Sequence Homology </li></ul><ul><li>Group I     (GroES dependent) GroEL        eubacteria Hsp60        mitochondria      Rubisco      chloroplasts </li></ul><ul><li>Group II    (GroES independent) thermosome/TF55     archaea TCP1/CCT               eukaryotic </li></ul>
  4. 4. Causes of Aggregations <ul><li>Hydrophobic Interactions </li></ul><ul><li>Interchain hydrogen bonding </li></ul><ul><li>Intracellular Crowding </li></ul><ul><li>U = Unfolded protein chain </li></ul><ul><li>N = Native fold protein </li></ul><ul><li>I = partially folded Intermediate </li></ul>
  5. 5. Consequences of Aggregation <ul><ul><li>Amyloid results from Structured fibrillar aggregates </li></ul></ul><ul><ul><li>Associated Diseases     Alzheimer's     Huntington's </li></ul></ul>
  6. 6. Chaperonins counteract non-native protein aggregation <ul><li>During de novo folding Under Stress (e.g. high temperature) </li></ul>
  7. 7. Experimental Details <ul><li>Method </li></ul><ul><ul><ul><li>X-Ray Diffraction </li></ul></ul></ul><ul><li>Resolution </li></ul><ul><ul><ul><li>3A </li></ul></ul></ul><ul><li>R-Factor </li></ul><ul><ul><ul><li>24.8% </li></ul></ul></ul><ul><li>Crystal Unit Cell Dimensions </li></ul><ul><ul><ul><li>dim [Å]: a 255.26 b 265.25 c 184.40 </li></ul></ul></ul><ul><ul><ul><li>angles [°]: alpha 90.00 beta 90.00 gamma 90.00 </li></ul></ul></ul><ul><li>Space Group </li></ul><ul><ul><ul><li>P2 1 2 1 2 </li></ul></ul></ul>
  8. 8. GroEL-GroES Architecture E = E. coli <ul><li>GroES </li></ul><ul><li>S = Small One heptameric ring 7 identical 10kD subunits </li></ul><ul><li>Chains O-U </li></ul><ul><li>GroEL L = Large </li></ul><ul><li>Two heptameric rings stacked back to back 14 identical 57kD subunits </li></ul><ul><li>Chains A-N </li></ul>
  9. 9. GroEL-GroES Architecture Equatorial = pink Intermediate = yellow Apical = blue Polar + Charge = blue Hydrophobic = yellow Backbone = white Solvent-Excluded surfaces = gray
  10. 10. GroES-GroEL Dimensions
  11. 11. GroEL-GroES Sequences 8337 Residues 58884 Atoms Equatorial = orange Intermediate = purple Apical = cyan
  12. 15. GroES Domain <ul><li>CATH </li></ul><ul><li>Mainly Beta Roll </li></ul>
  13. 16. GroEL Domains <ul><li>CATH </li></ul><ul><li>Equatorial </li></ul><ul><li>Mainly Alpha Orthogonal Bundle </li></ul><ul><li>Intermediate </li></ul><ul><li>Alpha Beta 2-Layer Sandwich </li></ul><ul><li>Apical </li></ul><ul><li>3-Layer(bba) Sandwich </li></ul>
  14. 21. GroES Mobile Loop
  15. 22. GroEL Domains
  16. 23. GroEL-GroES Binding Sites
  17. 24. Conformational Change
  18. 25. Conformational Change
  19. 26. Conformational Change of Cavity
  20. 27. Conformational Change
  21. 28. How do chaperonins work? <ul><li>Bind non-native polypeptide through hydrophobic interaction </li></ul><ul><li>Allow non-native polypeptide to fold in an isolated hydrophobic environment </li></ul>
  22. 29. Overall Chaperonins Protein Folding Reaction <ul><li>1. Non-native polypeptide bind to trans ring of GroEL 2. 7ATP (equatorial) and GroES bind cis ring of GroEL 2. Dissociation of 7ADP and GroES from from cis ring of GroEL 3. Apical domain of GroEL undergo massive rotation and upward movement enlarging the cavity by 2X and shifting its surface properties from hydropobic to hydrophilic </li></ul>
  23. 30. <ul><li>E. Coli have 4300 proteins </li></ul><ul><li>13% are ~55kD (500 residues) </li></ul><ul><li>10% polypeptides transit </li></ul><ul><li>GroEL-GroES complex </li></ul><ul><li>~3uM cytosolic concentration of GroEL </li></ul><ul><li>~30uM ribosomes </li></ul><ul><li>½ life of 20-60 kD folded proteins 15sec to few mins </li></ul>
  24. 31. 1AON chain A = red 1A6E chain A = yellow 1A6D chain A = green 1A6E Thermosome - Mg-ADP-Alf3 Complex 1A6D Thermosome From T. Acidophilum Ca 4.15A 87 atoms Ca 4.58 A 87 atoms
  25. 32. Structural Neighbors <ul><li>Criteria used: </li></ul><ul><li>1AON chain A </li></ul><ul><li>Z-Score>4.0 </li></ul><ul><li>RMSD<4.0Å </li></ul><ul><li>Length Difference<30.0% </li></ul><ul><li>Gaps<30.0% </li></ul><ul><li>Sequence identity<30.0% </li></ul><ul><li>Found & used: 1BPW:A </li></ul><ul><li>Z-Score 4.2 </li></ul><ul><li>RMSD(Å) 3.8 </li></ul><ul><li>Seq.(%) 2.3 </li></ul><ul><li>Aligned / Size 88 / 503 </li></ul><ul><li>Gap 25 </li></ul><ul><li>Exp X-Ray </li></ul>