High pressure spectrofluorimetry – a tool to determine protein-ligand binding volume

The change in protein volume observed upon protein-ligand interaction (termed as the binding volume) is an important but largely neglected thermodynamic parameter from the perspective of both fundamental science and potential applications in the development of specific protein ligands. The binding volume is the pressure derivative of the Gibbs energy, thus elevated pressure is required to determine the volumetric properties of proteins. Here we describe the use of high-pressure spectrofluorimetry to determine both unfolding and ligand binding- induced volume changes of a protein. The degree of protein unfolding at elevated pressures was monitored by an intrinsic tryptophan fluorescence. Different approaches of experimental fluorescence spectra analysis are described and the impact on the quality of thermodynamic parameters is discussed.