Determination of the Volume Changes Induced by Ligand Binding to Hsp90 Using High Pressure Denaturation

Abstract

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).Increased concentration of a ligand caused the shift in the Pm as shown in the Figure. The volumes of the protein in the absence and presence of several ligands were determined. The compressibilities of the system could be also determined with significantly lower precision than the volumes. The formulas describing the relationship between the added ligand concentration and the Pm were derived and will be presented.View Large Image | View Hi-Res Image | Download PowerPoint Slide