Wallace L A, Sluis-Cremer N, Dirr H W
Department of Biochemistry, University of the Witwatersrand, Johannesburg 2050, South Africa.
Biochemistry. 1998 Apr 14;37(15):5320-8. doi: 10.1021/bi972936z.
The equilibrium and kinetic unfolding properties of homodimeric class alpha glutathione transferase (hGST A1-1) were characterized. Urea-induced equilibrium unfolding data were consistent with a folded dimer/unfolded monomer transition. Unfolding kinetics were investigated, using stopped-flow fluorescence, as a function of denaturant concentration (3.5-8.9 M urea) and temperature (10-40 degrees C). The unfolding pathway, monitored by tryptophan fluorescence, was biphasic with a fast unfolding event (millisecond time range with enhanced fluorescence properties) and a slow unfolding event (seconds to minutes time range with quenched fluorescence properties). Both events occurred simultaneously from 3.5 M urea. Each phase displayed single-exponential behavior, consistent with two unimolecular reactions. Urea-dependence studies and thermodynamic activation parameters (transition-state theory) suggest that the transition state for each phase is well-structured and is closely related to native protein in terms of solvent exposure. The apparent activation Gibbs free energy change in the absence of denaturant, DeltaG (H2O), indicates that the slow unfolding event represents the transition state for the overall unfolding pathway. The rate and urea independence of each phase on the initial condition exclude the possibility of a preexisting equilibrium between various native forms in the pretransition baseline. The unfolding pathways monitored by energy transfer to or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding event (described above). A sequential unfolding kinetic mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed.
对同二聚体α类谷胱甘肽转移酶（hGST A1-1）的平衡和动力学去折叠特性进行了表征。尿素诱导的平衡去折叠数据与折叠二聚体/未折叠单体转变一致。使用停流荧光法研究了去折叠动力学，该动力学是变性剂浓度（3.5 - 8.9 M尿素）和温度（10 - 40℃）的函数。通过色氨酸荧光监测的去折叠途径是双相的，有一个快速去折叠事件（毫秒时间范围，荧光特性增强）和一个缓慢去折叠事件（秒到分钟时间范围，荧光特性淬灭）。从3.5 M尿素开始，这两个事件同时发生。每个阶段都表现出单指数行为，与两个单分子反应一致。尿素依赖性研究和热力学活化参数（过渡态理论）表明，每个阶段的过渡态结构良好，并且在溶剂暴露方面与天然蛋白质密切相关。在没有变性剂的情况下，表观活化吉布斯自由能变化ΔG（H₂O）表明，缓慢去折叠事件代表了整个去折叠途径的过渡态。每个阶段对初始条件的速率和尿素独立性排除了在转变前基线中各种天然形式之间预先存在平衡的可能性。通过能量转移到与hGST A1-1中Cys111共价连接的AEDANS或直接激发AEDANS来监测的去折叠途径是单相的，其尿素和温度特性与上述缓慢去折叠事件观察到的相似。提出了一种顺序去折叠动力学机制，该机制涉及每个亚基的两个结构不同结构域的部分解离，随后是结构域和亚基的完全去折叠。
