The equilibrium unfolding of hen lysozyme at pH 2 was studied as a function of pressure (0.1~700 MPa) and temperature (−10°C~50°C) using Trp fluorescence as monitor supplemented by variable pressure
1H NMR spectroscopy (0.1~400 MPa). 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
G (=
GU−
GN) were evaluated from changes in the wavelength of maximum fluorescence intensity (
λmax) as a function of pressure and temperature. The dependence of Δ
G on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (Δ
Cp=
CpU−
CpN=1.8−1.9 kJ mol
–1 deg
–1) at all pressures studied (250~400 MPa), while the temperature
TS for maximal Δ
G increased from about 10°C at 250 MPa to about 40°C at 550 MPa. The dependence of Δ
G on pressure gave negative volume changes (Δ
V=
VU−
VN) upon unfolding at all temperatures studied (−86~−17 ml mol
–1 for −10°C~50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δ
α~1.07 ml mol
–1 deg
–1). A phase-diagram between N and U (for Δ
G=0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (Δ
G) is presented on the
p-T plane.
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