Desai G, Panick G, Zein M, Winter R, Royer C A
School of Pharmacy, University of Wisconsin-Madison, 425 N Charter, Madison, WI, 53706, USA.
J Mol Biol. 1999 May 7;288(3):461-75. doi: 10.1006/jmbi.1999.2692.
The dimeric protein, trp apo-repressor of Escherichia coli has been subjected to high hydrostatic pressure under a variety of conditions, and the effects have been monitored by fluorescence spectroscopic and infra-red absorption techniques. Under conditions of micromolar protein concentration and low, non-denaturing concentrations of guanidinium hydrochloride (GuHCl), tryptophan and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence detected high pressure profiles demonstrate that pressures below 3 kbar result in dissociation of the dimer to a monomeric species that presents no hydrophobic binding sites for ANS. The FTIR-detected high pressure profile obtained under significantly different solution conditions (30 mM trp repressor in absence of denaturant) exhibits a much smaller pressure dependence than the fluorescence detected profiles. The pressure-denatured form obtained under the FTIR conditions retains about 50 % alpha-helical structure. From this we conclude that the secondary structure present in the high pressure state achieved under the conditions of the fluorescence experiments is at least as disrupted as that achieved under FTIR conditions. Fluorescence-detected pressure-jump relaxation studies in the presence of non-denaturing concentrations of GuHCl reveal a positive activation volume for the association/folding reaction and a negative activation volume for dissociation/unfolding reaction, implicating dehydration as the rate-limiting step for association/folding and hydration as the rate-limiting step for unfolding. The GuHCl concentration dependence of the kinetic parameters place the transition state at least half-way along the reaction coordinate between the unfolded and folded states. The temperature dependence of the pressure-jump fluorescence-detected dissociation/unfolding reaction in the presence of non-denaturing GuHCl suggests that the curvature in the temperature dependence of the stability arises from non-Arrhenius behavior of the folding rate constant, consistent with a large decrease in heat capacity upon formation of the transition state from the unfolded state. The decrease in the equilibrium volume change for folding with increasing temperature (due to differences in thermal expansivity of the folded and unfolded states) arises from a decrease in the absolute value for the activation volume for unfolding, thus indicating that the thermal expansivity of the transition state is similar to that of the unfolded state.
大肠杆菌的二聚体蛋白色氨酸脱辅基阻遏物在多种条件下经受了高静水压，其影响通过荧光光谱和红外吸收技术进行监测。在微摩尔蛋白浓度以及低浓度、非变性浓度的盐酸胍（GuHCl）条件下，色氨酸和8-苯胺基-1-萘磺酸盐（ANS）荧光检测到的高压图谱表明，低于3千巴的压力会导致二聚体解离为单体形式，该单体形式不存在与ANS的疏水结合位点。在显著不同的溶液条件下（30 mM色氨酸阻遏物，无变性剂）获得的傅里叶变换红外光谱（FTIR）检测到的高压图谱显示，其压力依赖性比荧光检测到的图谱小得多。在FTIR条件下获得的压力变性形式保留了约50%的α-螺旋结构。由此我们得出结论，在荧光实验条件下达到的高压状态下存在的二级结构至少与在FTIR条件下达到的一样受到破坏。在非变性浓度的GuHCl存在下进行的荧光检测压力跳跃弛豫研究表明，缔合/折叠反应的活化体积为正，解离/展开反应的活化体积为负，这意味着脱水是缔合/折叠的限速步骤，水合是展开的限速步骤。动力学参数对GuHCl浓度的依赖性表明，过渡态至少位于未折叠态和折叠态之间反应坐标的中间位置。在非变性GuHCl存在下，压力跳跃荧光检测到的解离/展开反应的温度依赖性表明，稳定性的温度依赖性中的曲率源于折叠速率常数的非阿累尼乌斯行为，这与从未折叠态形成过渡态时热容量的大幅降低一致。随着温度升高，折叠平衡体积变化的减小（由于折叠态和未折叠态热膨胀系数的差异）源于展开活化体积绝对值的减小，因此表明过渡态的热膨胀系数与未折叠态相似。
