The pressure-temperature phase diagram of hen lysozyme at low pH

Abstract

The equilibrium unfolding of hen lysozyme at pH 2 was studied as a function of pressure (0.1~700MPa) and temperature (-10°C~50°C) using Trp fluorescence as monitor supplemented by variable pressure ¹H NMR spectroscopy (0.1~400MPa). The unfolding profiles monitored by the two methods allowed the two-state equilibrium analysis between the folded (N) and unfolded (U) conformers. The free energy differences Δ<i>G</i> (=<i>G</i>_U-<i>G</i>_N) were evaluated from changes in the wavelength of maximum fluorescence intensity (<i>λ</i>_max) as a function of pressure and temperature. The dependence of Δ<i>G</i> on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (Δ<i>C</i><i>_p</i> =<i>C</i><i>_p</i>_U-<i>C</i><i>_p</i>_N= 1.8-1.9 kJ mol^-1 deg^-1) at all pressures studied (250~400 MPa), while the temperature <i>T</i>_S for maximal Δ<i>G</i> increased from about 10°C at 250MPa to about 40°C at 550MPa. The dependence of Δ<i>G</i> on pressure gave negative volume changes (Δ<i>V</i>=<i>V</i>_U-<i>V</i>_N) upon unfolding at all temperatures studied (-86~-17 mlmol^-1 for -10°C~50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δ<i>α</i>~1.07mlmol^-1 deg^-1). A phase-diagram between N and U (for Δ<i>G</i>=0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (Δ<i>G</i>) is presented on the <i>p-T</i> plane.