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Evidence for Weakened Activity of Mycobacterial Chaperonins

Evidence for Weakened Activity of Mycobacterial Chaperonins. C. M. Santosh Kumar, Garima Khare , C. V. Srikanth , Anil K Tyagi , Abhijit A. Sardesai and Shekhar C. Mande. Activity Restoration of a Naturally Inactive Chaperonin via Facilitated Oligomerization.

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Evidence for Weakened Activity of Mycobacterial Chaperonins

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  1. Evidence for Weakened Activity of Mycobacterial Chaperonins C. M. Santosh Kumar,GarimaKhare, C. V. Srikanth, Anil K Tyagi, Abhijit A. Sardesai and Shekhar C. Mande Activity Restoration of a Naturally Inactive Chaperonin via Facilitated Oligomerization Centre for DNA Fingerprinting and Diagnostics, Bldg. 7, Gruhakalpa, 5-4-399 / B, Nampally,  Hyderabad - 500 001 

  2. GroEL-GroES Chaperonin Complex • Best studied molecular chaperones • Two proteins – GroEL & GroES • GroEL - Tetradecamer of 58 kDasubunits • Two isologusheptameric rings • Hydrophobic cavities bind protein substrates • ATP dependent • GroES- Heptamer of 10 kDa, acts as a lid GroES GroEL ~ 10% of newly synthesized proteins

  3. Unusual GroELsof M. tuberculosis GroEL of M. tuberculosis • Two copies cpn60s: Mtb groEL1(cpn60.1) & Mtb groEL2(cpn60.2) • Share 70% homology with E. coli counterpart, GroEL • Dimeric form & ATP independent • Cannot help refolding but prevent aggregation GroEL Qamra and Mande., J. Bact. (2004) Qamra, Srinivas and Mande., JMB (2004) Cpn60.2

  4. Substrate Recognition or Oligomerization ?

  5. In vivo Complementation Studies RBS PgroE E. coli groES E. coli groEL • MtbgroEL1& Mtb groEL2cloned along with cohort MtbgroES • E. coli SV2 (groEL Ts mutant) and LG6 (GroES/L depletion strain) MtbGroES + GroEL1 MtbGroES + GroEL2 E. coli GroES + GroEL Vector RBS Plac E. coli groES MG1655 LG6 E. coli groEL • MtbgroELsare expressed but not able to complement

  6. Gene Shuffling E. coli SV2 0.2 % L-arabinose Incubated at 30 ℃and 42 ℃

  7. Sequence Analysis of Gene Shuffled Mutants • Apical domain is plastic & equatorial domain is conserved

  8. Impaired Oligomerization • – Activity Determinant Apical Domain Intermediate Domain Equatorial Domain • Equatorial Domains Exchanged

  9. Complementation BacteriophagePlaquing Bacteriophage SV2 Lawn 0.2 % L-arabinose Incubated at 30 ℃

  10. RBS PgroE E. coli groES E. coli groEL Resident GroEL Contamination? MG1655 MGM100 RBS PBAD E. coli groES E. coli groEL • Ptac based vector • GroELMEFactive in vivo – GroES dependent

  11. Protein Purification E. coli GroEL Expressed in E. coli BL21 (DE3) Auto Induction System or L-arabinose Ammonium Sulphate Extraction Ion Exchange or Hydrophobic Interaction Chromatography Gel Filtration GroELMEF GroELMER GroELSp24 GroELSp32 • GroELMEF exists as oligomer

  12. Biochemical Characterization ATPaseActivity Substrate Protein Refolding Prevention of Aggregation

  13. Summary • MtbrGroEL - Inherently Inactive Chaperones • GroELSp24 & GroELSp32 • Efficiency of Substrate Binding • GroELMEF - Functional Chaperone • Complementation • Exists as Oligomer - Refolding & ATPase Activity • Stability • GroELMER - Inactive as Chaperone • Prevents Substrate Protein Aggregation • Impaired Oligomerization of MtbrGroELs – Reduced Activity

  14. Factor Mediated Oligomerization? • Active ATPase • Slow Growing Mtb – 24 hrs doubling time • CanMtb Tolerate Robust GroEL? • Residues mutated to Serine or Threonine • Eukaryotic like STPKs • Heat regulated oligomerization in chaperones • Heat or Phosphorylation?

  15. What Forms in Mtb? Mtblysates resolved by Gel filtration Superdex S200 16/60 Mtblysates resolved on Native PAGE Native PAGE SDS PAGE Probed with α-GroEL1 antibody Peaks corresponding to different Oligomeric forms of GroEL Normallized fraction of each peak probed - α-GroEL1 antibody 37 ℃ 42 ℃ • GroEL1 predominates in three forms

  16. Which Form of GroEL? Is GroEL Phosphorylated? Co-immuno Precipitation Western Blotting Peaks corresponding to different Oligomeric forms of GroEL α-Ser-P antibody • Tetradecameric form is Phosphorylated on Serine residue

  17. Which Serine? Mass-Spec Analysis Acrylamide plugs (Oligomeric forms of GroEL) MALDI-TOF MS/MS Peptides with serine P ESVEDAVAAAK • S393 is Phosphorylated

  18. Summary • Mycobacterial GroEL1 Exists in Multiple Forms • Heptameric Form - Ambient Conditions • Tetra-decameric Form - Elevated Temperatures • Phosphorylation • S393 is Phosphorylated – No Threonine • Switch Between Heptameric & Tetra-decameric Form • GroEL2 is not phosphorylated! • Oligomerization (Activity) is Phosphorylation Mediated

  19. The Model Mitochondrial & Choloroplast Hsp60 Levy et. al., Eur. J. Biochem. 2001 Dickson et. al., J. Biol. Chem. 2000

  20. ACKNOWLEDGEMENTS • CDFD, Hyderabad • Department of Biotechnology, India • Council of Scientific and Industrial Research, India • Wellcome Trust, UK

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