title: 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 pressure1H 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(=GU-GN) were evaluated from changes in the wavelength of maximum fluorescence intensity (λmax) as a function of pressure and temperature. The dependence of ΔGon temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (ΔCp=CpU-CpN= 1.8-1.9 kJ mol-1deg-1) at all pressures studied (250~400 MPa), while the temperatureTSfor maximal ΔGincreased from about 10°C at 250MPa to about 40°C at 550MPa. The dependence of ΔGon pressure gave negative volume changes (ΔV=VU-VN) upon unfolding at all temperatures studied (-86~-17 mlmol-1for -10°C~50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δα~1.07mlmol-1deg-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 thep-Tplane.
authors.affiliation: Graduate School of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan; RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Maeno
authors.affiliation: Niigata Industrial Creation Organization, 5-1 Bandaijima, Chuo-ku, Niigata 950-0078, Japan; High Pressure Protein Research Center, Institute of Advanced Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuo
authors.affiliation: Graduate School of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan; RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; High Pressure Protein Research Center, Institute of Advanced Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Akasaka
keywords: free energy landscape; hen lysozyme; high pressure NMR; high pressure fluorescence; thermodynamic stability on pressure-temperature axes