Bacterial Hsp90 ATPase Assays

Joel R Hoskins; Sue Wickner; Shannon M Doyle

doi:10.1007/978-1-4939-7477-1_15

Bacterial Hsp90 ATPase Assays

Methods Mol Biol. 2018:1709:199-207. doi: 10.1007/978-1-4939-7477-1_15.

Authors

Joel R Hoskins¹, Sue Wickner², Shannon M Doyle³

Affiliations

¹ Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 5144, NIH, Bethesda, MD, 20892, USA.
² Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 5144, NIH, Bethesda, MD, 20892, USA. wickners@mail.nih.gov.
³ Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 5144, NIH, Bethesda, MD, 20892, USA. doyles@mail.nih.gov.

Abstract

Bacterial Hsp90 is an ATP-dependent molecular chaperone involved in protein remodeling and activation. The E. coli Hsp90, Hsp90_Ec, collaborates in protein remodeling with another ATP-dependent chaperone, DnaK, the E. coli Hsp70. Both Hsp90_Ec and DnaK hydrolyze ATP and client (substrate) proteins stimulate the hydrolysis. Additionally, ATP hydrolysis by the combination of Hsp90_Ec and DnaK is synergistically stimulated in the presence of client (substrate). Here, we describe two steady-state ATPase assays used to monitor ATP hydrolysis by Hsp90_Ec and DnaK as well as the synergistic stimulation of ATP hydrolysis by the combination of Hsp90_Ec and DnaK in the presence of a client (substrate). The first assay is a spectrophotometric assay based on enzyme-coupled reactions that utilize the ADP formed during ATP hydrolysis to oxidize NADH. The second assay is a more sensitive method that directly quantifies the radioactive inorganic phosphate released following the hydrolysis of [γ-³³P] ATP or [γ-³²P] ATP.

Keywords: ATP hydrolysis; DnaK; Hsp70; Hsp90; Steady-state ATPase.

MeSH terms

Adenosine Triphosphatases / analysis
Adenosine Triphosphatases / metabolism
Enzyme Assays / methods*
Escherichia coli / enzymology
Escherichia coli / metabolism*
Escherichia coli Proteins / analysis
Escherichia coli Proteins / metabolism*
HSP70 Heat-Shock Proteins / analysis
HSP70 Heat-Shock Proteins / metabolism*
HSP90 Heat-Shock Proteins / analysis
HSP90 Heat-Shock Proteins / metabolism*
Kinetics

Substances

Escherichia coli Proteins
HSP70 Heat-Shock Proteins
HSP90 Heat-Shock Proteins
HtpG protein, E coli
Adenosine Triphosphatases
dnaK protein, E coli

Grants and funding

ZIA BC008710/ImNIH/Intramural NIH HHS/United States