Brandts J F, Halvorson H R, Brennan M
Biochemistry. 1975 Nov 4;14(22):4953-63. doi: 10.1021/bi00693a026.
A model is proposed to account for the observation that the denaturation of small proteins apparently occurs in two kinetic phases. It is suggested that only one of these phases--the fast one--is actually an unfolding process. The slow phase is assumed to arise from the cis-trans isomerism of proline residues in the denaturated protein. From model compound data, it is shown that the expected rate for isomerism is in satisfactory agreement with the rates actually observed for protein folding. It is also shown that a simple model of protein unfolding based on the isomerism concept is very successful in accounting for many known experimental characteristics of the kinetics and thermodynamic of protein denaturation. Thus, the model is able to predict that two kinetic phases will be seen in the transition region while none are seen in the base-line regions, that both the fast and slow refolding phases lead to the native protein as the product, that the fast phase becomes the only observable phase for jumps ending far in the denatured base-line region, that most or all small proteins show a limiting low-temperature activation energy of ca. 20,000 cal, and that the relaxtion time for the slow phase seen in cytochrome c denaturation is much shorter than for all other small proteins. By utilizing "double-jump" experiments, it is shown directly that the slow phase is not part of the unfolding process but that it corresponds to a transition among two or more denatured forms which have identical spectroscopic (286.5 nm) properties. Thus, the slow relaxation is "invisible" except in the transition region where it couples to the fast unfolding equilibrium. Finally, since the present model assumes that only one of the major kinetic phases seen in denaturation reactions is concerned with the denaturation process per se, it is in agreement with numerous thermodynamic studies which show consistency with the two-state model for unfolding.
提出了一个模型来解释小蛋白质变性明显发生在两个动力学阶段的观察结果。有人认为，这些阶段中只有一个——快速阶段——实际上是一个展开过程。慢阶段被认为是由变性蛋白质中脯氨酸残基的顺反异构化引起的。从模型化合物数据可知，异构化的预期速率与蛋白质折叠实际观察到的速率令人满意地一致。还表明，基于异构化概念的简单蛋白质展开模型在解释蛋白质变性动力学和热力学的许多已知实验特征方面非常成功。因此，该模型能够预测在转变区域会看到两个动力学阶段，而在基线区域则看不到；快速和慢速重折叠阶段都以天然蛋白质为产物；对于在变性基线区域深处结束的跃迁，快速阶段成为唯一可观察到的阶段；大多数或所有小蛋白质显示出约20,000卡的极限低温活化能；细胞色素c变性中看到的慢阶段的弛豫时间比所有其他小蛋白质的弛豫时间短得多。通过利用“双跃迁”实验，直接表明慢阶段不是展开过程的一部分，而是对应于具有相同光谱（286.5纳米）性质的两种或更多种变性形式之间的转变。因此，慢弛豫是“不可见的”，除非在它与快速展开平衡耦合的转变区域。最后，由于目前的模型假设变性反应中看到的主要动力学阶段中只有一个与变性过程本身有关，所以它与许多热力学研究一致，这些研究表明与展开的两态模型一致。
