Bhuyan A K, Udgaonkar J B
National Centre for Biological Sciences, TIFR Centre, Indian Institute of Science, Bangalore.
Biochemistry. 1998 Jun 23;37(25):9147-55. doi: 10.1021/bi980470u.
The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degreesC. In contrast, no burst phase change in fluorescence occurs during unfolding at 10 degreesC. At 22 and 34 degreesC, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, IU1, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, IU2, has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22 degreesC. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of IU1 and IU2 decrease steadily with an increase in temperature from 10 to 34 degreesC, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions.
已在10℃、22℃和34℃下研究了氧化态马细胞色素c的展开动力学，该动力学是盐酸胍（GdnHCl）浓度的函数。利用远紫外圆二色性（CD）的快速（毫秒级）测量以及色氨酸到血红素激发能量转移导致的荧光猝灭来监测展开过程。在10℃时，伴随展开的远紫外CD信号下降分两个阶段发生。不可观察的猝发阶段在4毫秒内完成，而较慢阶段发生在数十到数百毫秒内。猝发阶段的展开幅度随着GdnHCl浓度的增加而协同增加，在10℃时表现出3.2 M的转变中点。相比之下，在10℃展开过程中荧光没有猝发阶段变化。在22℃和34℃时，荧光监测的展开动力学和远紫外CD监测的展开动力学都是双相的。在这两个温度下，两种探针产生的猝发阶段展开转变在转变中点以及猝发阶段幅度对GdnHCl浓度的依赖性方面都不一致。结果表明，在展开过程中至少积累了两种动力学展开中间体。一种猝发阶段中间体IU1几乎失去了所有天然状态的二级结构，而另一种猝发阶段中间体IU2则失去了二级结构和类似天然的紧密性。表观展开速率常数的对数对GdnHCl浓度的非线性依赖性也表明存在动力学展开中间体，这在10℃和22℃时尤为明显。对使用两种探针获得的猝发阶段展开转变的分析表明，随着温度从10℃升高到34℃，IU1和IU2的稳定性稳步下降，这表明它们中存在的结构主要通过氢键相互作用得以稳定。
