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Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
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Bacterial Hsp90 ATPase Assays.

Joel R Hoskins1, Sue Wickner2, Shannon M Doyle3

  • 1Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 5144, NIH, Bethesda, MD, 20892, USA.

Methods in Molecular Biology (Clifton, N.J.)
|November 28, 2017
PubMed
Summary

Bacterial heat shock protein 90 (Hsp90) and DnaK (Hsp70) work together to remodel proteins. Their combined ATP hydrolysis is synergistically stimulated by client proteins, as measured by two novel ATPase assays.

Keywords:
ATP hydrolysisDnaKHsp70Hsp90Steady-state ATPase

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Area of Science:

  • Molecular biology
  • Biochemistry
  • Protein folding

Background:

  • Bacterial Hsp90 (heat shock protein 90) is a crucial ATP-dependent molecular chaperone.
  • It collaborates with other chaperones, such as E. coli Hsp70 (DnaK), in protein remodeling and activation.
  • Both Hsp90 and DnaK hydrolyze ATP, a process stimulated by client proteins.

Purpose of the Study:

  • To describe two steady-state ATPase assays for monitoring ATP hydrolysis by Hsp90Ec and DnaK.
  • To characterize the synergistic stimulation of ATP hydrolysis by the Hsp90Ec-DnaK complex in the presence of client proteins.

Main Methods:

  • A spectrophotometric assay utilizing enzyme-coupled reactions to monitor NADH oxidation, linked to ADP formation from ATP hydrolysis.
  • A radioactive assay for direct quantification of inorganic phosphate released from [γ-33P] ATP or [γ-32P] ATP hydrolysis.

Main Results:

  • The study details methodologies for measuring ATP hydrolysis by individual chaperones and their complex.
  • Demonstrated synergistic stimulation of ATP hydrolysis by the Hsp90Ec-DnaK combination upon client protein binding.
  • Established reliable assays for studying chaperone-mediated protein remodeling dynamics.

Conclusions:

  • Developed and validated two distinct ATPase assays for bacterial Hsp90 and DnaK.
  • The assays enable quantitative analysis of chaperone activity and client-induced modulation.
  • These methods facilitate further investigation into the mechanistic details of chaperone collaboration in protein homeostasis.