Journal
ANALYTICAL BIOCHEMISTRY
Volume 413, Issue 2, Pages 171-178Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.02.019
Keywords
Thermal shift assay; ThermoFluor; Pressure shift assay; PressureFluor; Hsp90; Ligand binding volume
Funding
- EEA and Norway [2004-LT0019-IP-1EEE]
- Lithuanian government
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The volume changes accompanying ligand binding to proteins are thermodynamically important and could be used in the design of compounds with specific binding properties. Measuring the volumetric properties could yield as much information as the enthalpic properties of binding. Pressure-based methods are significantly more laborious than temperature methods and are underused. Here we present a pressure shift assay (PressureFluor, analogous to the ThermoFluor thermal shift assay) that uses high pressure to denature proteins. The PressureFluor method was used to study the ligand binding thermodynamics of heat shock protein 90 (Hsp90). Ligands stabilize the protein against pressure denaturation, similar to the stabilization against temperature denaturation. The equations that relate the ligand dosing, protein concentration, and binding constant with the volumes and compressibilities of unfolding and binding are presented. (C) 2011 Elsevier Inc. All rights reserved.
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