Enoki Sawako, Maki Kosuke, Inobe Tomonao, Takahashi Kazunobu, Kamagata Kiyoto, Oroguchi Tomotaka, Nakatani Hiroyasu, Tomoyori Katsuaki, Kuwajima Kunihiro
Department of Physics, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
J Mol Biol. 2006 Sep 1;361(5):969-82. doi: 10.1016/j.jmb.2006.07.009. Epub 2006 Aug 4.
Folding mechanisms of a variant of green fluorescent protein (F99S/M153T/V163A) were investigated by a wide variety of spectroscopic techniques. Equilibrium measurements on acid-induced denaturation of the protein monitored by chromophore and tryptophan fluorescence and small-angle X-ray scattering revealed that this protein accumulates at least two equilibrium intermediates, a native-like intermediate and an unfolding intermediate, the latter of which exhibits the characteristics of the molten globule state under moderately denaturing conditions at pH 4. To elucidate the role of the equilibrium unfolding intermediate in folding, a series of kinetic refolding experiments with various combinations of initial and final pH values, including pH 7.5 (the native condition), pH 4.0 (the moderately denaturing condition where the unfolding intermediate is accumulated), and pH 2.0 (the acid-denaturing condition) were carried out by monitoring chromophore and tryptophan fluorescence. Kinetic on-pathway intermediates were accumulated during the folding on the refolding reaction from pH 2.0 to 7.5. However, the signal change corresponding to the conversion from the acid-denatured to the kinetic intermediate states was significantly reduced on the refolding reaction from pH 4.0 to pH 7.5, whereas only the signal change corresponding to the above conversion was observed on the refolding reaction from pH 2.0 to pH 4.0. These results indicate that the equilibrium unfolding intermediate is composed of an ensemble of the folding intermediate species accumulated during the folding reaction, and thus support a hierarchical model of protein folding.
通过多种光谱技术研究了绿色荧光蛋白变体（F99S/M153T/V163A）的折叠机制。通过发色团和色氨酸荧光以及小角X射线散射监测蛋白质酸诱导变性的平衡测量结果表明，该蛋白质积累了至少两种平衡中间体，一种类似天然态的中间体和一种去折叠中间体，后者在pH 4的适度变性条件下表现出熔球态的特征。为了阐明平衡去折叠中间体在折叠过程中的作用，通过监测发色团和色氨酸荧光，进行了一系列初始和最终pH值不同组合的动力学重折叠实验，包括pH 7.5（天然条件）、pH 4.0（积累去折叠中间体的适度变性条件）和pH 2.0（酸变性条件）。在从pH 2.0到7.5的重折叠反应中，折叠过程中积累了动力学途径中间体。然而，在从pH 4.0到pH 7.5的重折叠反应中，对应于从酸变性态到动力学中间体态转变的信号变化显著降低，而在从pH 2.0到pH 4.0的重折叠反应中仅观察到对应于上述转变的信号变化。这些结果表明，平衡去折叠中间体由折叠反应过程中积累的折叠中间体物种的集合组成，因此支持蛋白质折叠的层级模型。
