Kinetics and mechanism of the folding of cytochrome c.
Kinetics and mechanism of the folding of cytochrome c.
复制标题
细胞色素c折叠的动力学和机制。
DOI：
10.1021/bi00137a006
复制
发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Elson,EL
中科院分区：
文献类型：
--
作者：
Pryse,KM;Bruckman,TG;Maxfield,BW;Elson,EL
1992年3月25日摘要：用重复压力扰动动力学和平衡光谱法研究了pH 4.0时细胞色素c在尿素中的可逆折叠。在1 ms至10 s的时间范围内观察到两个折叠反应。这些反应的速率和幅度以复杂的方式取决于尿素浓度，这对于每个过程是不同的。两个反应的动力学振幅的吸收光谱也彼此不同。一个三态机制的模型可以定量地解释所有的动力学和平衡数据，它使我们能够确定的速率常数和体积变化的两个步骤。如果一个快速的质子化步骤被添加到该机制，分析可以扩展到计算的速度和幅度的pH值依赖性的更快的折叠步骤。这种pH依赖性与先前公布的数据非常一致[Tsong，TY（1977）J.Biol.Chem.252，8778-8780]。在695-nm波段的动力学实验清楚地表明，轴向配体蛋氨酸-80参与缓慢折叠过程，而另一个轴向配体组氨酸-18参与快速过程。其他实验与溴化氰fragment的蛋白质，和荧光检测的折叠动力学的完整蛋白质，支持解释的模型中已知的结构元素的细胞色素c。这项工作为细胞色素c的折叠机制提供了新的信息，解决了早期解释中的冲突，并证明了重复压力扰动动力学方法对蛋白质折叠的适用性。
Revised Manuscript Received March 25, 1992 abstract: The reversible folding of cytochrome c in urea at pH 4.0 was investigated by repetitive pressure perturbation kinetics and by equilibrium spectroscopic methods. Twofolding reactions were observed in the 1 ms to 10 s time range. The rates and amplitudes of these reactions depend on urea concentration in a complex manner, which is different for each process. The absorbance spectra of the kinetic amplitudes of the two reactions also differ from each other. A model with a three-state mechanism can quantitatively account for all of the kinetic and equilibrium data, and it enables us to determine the rate constants and volume changes of the two steps. If a rapid protonation step is added to the mechanism, the analysis can be extended to calculate the pH dependence of the rate and amplitude of the faster folding step. This pH dependence is in excellent agreement with previously published data [Tsong, TY (1977) J. Biol. Chem. 252, 8778-8780]. Kinetic experiments in the 695-nm band show clearly that the axial ligand methionine-80 is involved in the slow folding process andthe other axial ligand, histidine-18, is involved in the fast process. Additional experiments with a cyanogen bromidefragment of the protein, and fluorescence detection of the folding kinetics of the intact protein, support an interpretation of the model in terms of known structural elements of cytochrome c. This work provides new information aboutthe mechanism of the folding of cytochrome c, resolves conflicts in earlier interpretations, and demonstrates the applicability of the repetitive pressure perturbation kinetics methodto protein folding.