title: Determination of the volume changes induced by ligand binding to heat shock protein 90 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). 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.
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania.
authors.forename: Zigmantas
authors.initials: Z
authors.lastname: Toleikis
authors.forename: Piotras
authors.initials: P
authors.lastname: Cimmperman
authors.forename: Vytautas
authors.initials: V
authors.lastname: Petrauskas
authors.forename: Daumantas
authors.initials: D
authors.lastname: Matulis
mesh_terms: D016227:Benzoquinones / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry*; D047029:Lactams, Macrocyclic / Q000737:chemistry*; D008024:Ligands; D018942:Macrolides / Q000737:chemistry*; D008968:Molecular Conformation; D011312:Pressure; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D058767:Protein Unfolding; D013816:Thermodynamics
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D008024:Ligands; D018942:Macrolides; C112765:tanespimycin; C035359:monorden; D019791:Guanidine