European Biophysics Journal

, Volume 42, Issue 5, pp 355–362 | Cite as

Volume of Hsp90 ligand binding and the unfolding phase diagram as a function of pressure and temperature

  • Vytautas Petrauskas
  • Joana Gylytė
  • Zigmantas Toleikis
  • Piotras Cimmperman
  • Daumantas Matulis
Original Paper


Volume changes that accompany protein unfolding and ligand binding are important but largely neglected thermodynamic parameters that may facilitate rational drug design. Here, we determined the volume of lead compound ICPD47 binding to an anticancer target, heat shock protein 90 N-terminal domain, using a pressure shift assay (PressureFluor). The ligand exhibited a stabilizing effect on the protein by increasing its melting pressure and temperature. The Gibbs free energy of unfolding depends on the absence or presence of ligand and has an elliptical shape. Ellipse size increases upon addition of the strongly binding ligand, which stabilizes the protein. The three-dimensional (3D) ellipsoidal surface of the Gibbs free energy of unfolding was calculated with increasing ligand concentrations. The negative volume of ligand binding was relatively large and significantly exceeded the volume of protein unfolding. The pressure shift assay technique could be used to determine the volume changes associated with both protein unfolding as well as ligand binding to protein.


Phase diagram Protein stability Thermal shift assay ThermoFluor® Pressure shift assay PressureFluor Hsp90N 



The authors thank Johnson & Johnson Pharmaceutical Research and Development for the donation of the ISS PC1 spectrofluorimeter, Cathy Royer for the donation of the hydrostatic pump, FP7-REGPOT-2009-1 grant "MoBiLi,” agreement no.: 245721, and the COST projects TD0905 and CM0804. P.C. and J.G. thank the European Social Fund and the Republic of Lithuania (grant numbers VP1-3.2-ŠMM-01-K-02-002 and VP1-3.1-ŠMM-01-V-01-002, respectively) for financial support.


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Copyright information

© European Biophysical Societies' Association 2013

Authors and Affiliations

  • Vytautas Petrauskas
    • 1
  • Joana Gylytė
    • 1
  • Zigmantas Toleikis
    • 2
  • Piotras Cimmperman
    • 3
  • Daumantas Matulis
    • 1
  1. 1.Department of Biothermodynamics and Drug DesignVilnius University Institute of BiotechnologyVilniusLithuania
  2. 2.Structural Biology and NMR Laboratory, Department of BiologyUniversity of CopenhagenCopenhagenDenmark
  3. 3.Baltic Institute of Advanced TechnologyVilniusLithuania

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