Vogl T, Jatzke C, Hinz H J, Benz J, Huber R
Institut für Physikalische Chemie, Westfalische Wilhelms-Universität Münster, Germany.
Biochemistry. 1997 Feb 18;36(7):1657-68. doi: 10.1021/bi962163z.
Conformational stability of the membrane-binding protein annexin V E17G has been determined by high-sensitivity differential scanning microcalorimetry (DSC) measurements and by isothermal, guanidinium hydrochloride (GdnHCl)-induced unfolding studies. Wild-type annexin V and the E17G mutant protein studied here are structurally almost identical. Therefore, it can be expected that the present results will not deviate significantly from the stability data of the wild-type molecule. Thermal unfolding is irreversible, while GdnHCl unfolding shows a high degree of reversibility. We were able to demonstrate that characteristic features of annexin V E17G unfolding permit us to extract from the kinetically controlled heat capacity curves thermodynamic equilibrium parameters at the high heating rates. The thermodynamic quantities obtained from the DSC studies in phosphate buffer at pH 7.0 are as follows: t1/2 = 54.7 degrees C (heating rate of 2.34 K min-1), delta H0 = 690 kJ mol-1, and delta Cp = 10.3 kJ mol-1 K-1 which correspondends to a value of delta G0D (20 degrees C) of 53.4 kJ mol-1. When compared on a per gram basis, these thermodynamic parameters classify annexin V E17G as a marginally stable protein. This conclusion is consistent with structural and functional features of the protein that require conformational adaptability for hinge-bending motions and pore formation on interaction with membranes. We observed a large difference between the change in the Gibbs energy value derived from the heat capacity studies and that determined from the GdnHCl unfolding curve. The difference appears to stem from a specific interaction of the protein with the denaturant that results in both a low half-denaturation concentration C1/2 of 1.74 M and a small slope (6.0 kJ L mol-2) of the delta Gapp versus [GdnHCl] plot. The extraordinary interaction of annexin V with GdnHCl is also manifested in the enormous depression of the transition temperature delta t1/2 (= 18 degrees C) when the GdnHCl concentration is increased from 0 to 1 M. "Regular" proteins experience an average decrease in the transition temperature of 8 +/- 2 degrees C per 1 M change in the concentration of GdnHCl.
膜结合蛋白膜联蛋白V E17G的构象稳定性已通过高灵敏度差示扫描量热法（DSC）测量以及等温、盐酸胍（GdnHCl）诱导的去折叠研究来确定。本文研究的野生型膜联蛋白V和E17G突变蛋白在结构上几乎相同。因此，可以预期目前的结果与野生型分子的稳定性数据不会有显著偏差。热去折叠是不可逆的，而GdnHCl去折叠显示出高度的可逆性。我们能够证明膜联蛋白V E17G去折叠的特征使我们能够在高加热速率下从动力学控制的热容曲线中提取热力学平衡参数。在pH 7.0的磷酸盐缓冲液中通过DSC研究获得的热力学量如下：t1/2 = 54.7℃（加热速率为2.34 K min-1），ΔH0 = 690 kJ mol-1，以及ΔCp = 10.3 kJ mol-1 K-1，这对应于20℃时ΔG0D的值为53.4 kJ mol-1。按每克计算时，这些热力学参数将膜联蛋白V E17G归类为一种稳定性略低的蛋白质。这一结论与该蛋白质的结构和功能特征一致，这些特征要求在与膜相互作用时具有构象适应性以进行铰链弯曲运动和形成孔。我们观察到从热容研究得出的吉布斯能量值变化与从GdnHCl去折叠曲线确定的值之间存在很大差异。这种差异似乎源于蛋白质与变性剂的特异性相互作用，这导致低的半变性浓度C1/2为1.74 M，以及ΔGapp对[GdnHCl]图的小斜率（6.0 kJ L mol-2）。当GdnHCl浓度从0增加到1 M时，膜联蛋白V与GdnHCl的特殊相互作用还表现为转变温度Δt1/2的大幅降低（= 18℃）。“常规”蛋白质在GdnHCl浓度每变化1 M时，转变温度平均降低8±2℃。
