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

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 (1)H 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 ΔG (=G(U)–G(N)) were evaluated from changes in the wavelength of maximum fluorescence intensity (λ(max)) as a function of pressure and temperature. The dependence of ΔG on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (ΔC(p)=C(p)(U)–C(p)(N)= 1.8–1.9 kJ mol(−1) deg(−1)) at all pressures studied (250~400 MPa), while the temperature T(S) for maximal ΔG increased from about 10°C at 250MPa to about 40°C at 550MPa. The dependence of ΔG on pressure gave negative volume changes (ΔV=V(U)–V(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 (Δα~1.07mlmol(−1) deg(−1)). A phase-diagram between N and U (for ΔG=0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (ΔG) is presented on the p-T plane.