Ghaemmaghami S, Word J M, Burton R E, Richardson J S, Oas T G
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 1998 Jun 23;37(25):9179-85. doi: 10.1021/bi980356b.
A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 micro(s) in the absence of denaturants. All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.
已制备出一种含色氨酸的单体λ阻遏物变体，并在20℃下使用荧光停流法和动态核磁共振对其折叠动力学进行了分析。通过圆二色性、荧光和核磁共振获得的平衡变性曲线是可叠加的。停流分析表明，在没有变性剂的情况下，折叠反应在实验的死时间内完成。在较高变性剂条件下，折叠速率较慢，核磁共振和停流法在蛋白质的折叠和解折叠速率上是一致的。在3.4 M尿素和1.8 M盐酸胍中，我们表明该变体在2毫秒内折叠。外推表明，在没有变性剂的情况下折叠时间为20微秒。所有的折叠和解折叠反应都表现出单指数动力学，并且没有观察到爆发相。此外，从动力学分析中获得的热力学参数ΔG和meq与平衡实验结果一致。这些结果支持了两态D⇌N折叠模型。
