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Acid denaturation and refolding of green fluorescent protein

机译：酸性变性和绿色荧光蛋白的折叠

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摘要

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.
机译：来自海母Aequorea victoria的绿色荧光蛋白可以作为很好的模型蛋白，以了解在复杂的环境中与分子伴侣和其他大分子（例如生物细胞中的分子）折叠的蛋白质，但是对这种蛋白质的体外折叠的详细机制了解甚少。绿色荧光蛋白本身。因此，我们研究了绿色荧光蛋白突变体（F99S / M153T / V163A）从酸变性状态的动力学重折叠，该突变体已知比野生型蛋白更有效地成熟。使用停止流技术，通过发色团荧光，色氨酸荧光和远紫外CD观察到重折叠。在这项研究中，我们证明了突变体重折叠的动力学至少具有五个动力学相，并且涉及到停流装置死时间内的非特异性塌陷，以及随后形成的具有中间体特征的途中中间体。熔融小球状态。我们还证明了最慢的相和第二最慢的相的主要部分受中间状态下缓慢的脯氨酰异构化速率限制，该速率限制占绿色荧光蛋白折叠中观察到的动力学的主要部分。

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