Acid denaturation and refolding of green fluorescent protein
Acid denaturation and refolding of green fluorescent protein
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DOI：
10.1021/bi048733
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发表时间：
2004-11-09
期刊：
影响因子：
2.9
通讯作者：
Kuwajima, K
中科院分区：
文献类型：
--
作者：
Enoki, S;Saeki, K;Kuwajima, K
水母（Aequorea victoria）的绿色荧光蛋白可以作为一种很好的模型蛋白，用于了解生物细胞中分子伴侣和其他大分子在复杂环境下的蛋白质折叠，但对绿色荧光蛋白自身在体外折叠的详细机制尚不清楚。因此，我们研究了绿色荧光蛋白突变体（F99S/M153T/V163A）的动力学重折叠，已知该突变体比野生型蛋白更有效地从酸变性状态成熟；通过发色团荧光、色氨酸荧光和远紫外CD，采用停流技术观察重折叠。在这项研究中，我们证明了突变体的再折叠动力学至少有五个动力学阶段，并且涉及在停止流动装置的死时间内的非特异性坍塌，以及随后形成具有熔融球态特征的通路中间体。我们还证明了最慢相和第二最慢相的大部分在中间状态受到缓慢的脯氨酸异构化的速率限制，这种速率限制是观察到的绿色荧光蛋白折叠动力学的主要部分。
Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.