Maeno Akihiro, Matsuo Hiroshi, Akasaka Kazuyuki
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.
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.
Biophysics (Nagoya-shi). 2009 Mar 11;5:1-9. doi: 10.2142/biophysics.5.1. eCollection 2009.
The equilibrium unfolding of hen lysozyme at pH 2 was studied as a function of pressure (0.1700MPa) and temperature (-10°C50°C) using Trp fluorescence as monitor supplemented by variable pressure H NMR spectroscopy (0.1400MPa). 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 (=-) were evaluated from changes in the wavelength of maximum fluorescence intensity () as a function of pressure and temperature. The dependence of Δ on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (Δ =-= 1.8-1.9 kJ mol deg) at all pressures studied (250400 MPa), while the temperature for maximal Δ increased from about 10°C at 250MPa to about 40°C at 550MPa. The dependence of Δ on pressure gave negative volume changes (Δ=-) upon unfolding at all temperatures studied (-86-17 mlmol for -10°C50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δ~1.07mlmol deg). A phase-diagram between N and U (for Δ=0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (Δ) is presented on the plane.
以色氨酸荧光为监测手段，并辅以可变压力氢核磁共振波谱法（0.1400MPa），研究了pH值为2时，压力（0.1700MPa）和温度（-10°C50°C）对溶菌酶平衡去折叠的影响。两种方法监测的去折叠曲线允许对折叠态（N）和去折叠态（U）构象之间的双态平衡进行分析。根据最大荧光强度波长（）随压力和温度的变化来评估自由能差Δ（=-）。Δ对温度的依赖性呈现出与温度的凹曲线，在所研究的所有压力（250400MPa）下均显示出正的热容变化（Δ=-=1.8-1.9kJ mol deg），而最大Δ对应的温度从250MPa时的约10°C增加到550MPa时的约40°C。在所有研究温度（-10°C50°C）下，Δ对压力的依赖性表明去折叠时体积变化为负（Δ=-），且随温度升高显著增加，呈现出正的膨胀系数变化（Δ1.07mlmol deg）。在压力-温度平面上绘制了pH值为2时溶菌酶的N态和U态之间的相图（Δ=0）。最后，在平面上展示了三维自由能景观（Δ）。
