Microsecond structural fluctuations in denatured cytochrome c and the mechanism of rapid chain contraction-猫眼课题宝

变性细胞色素c的微秒级结构波动及快速链收缩机制

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发表时间：

2003

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作者：

C. Rischel;Lars Elkjær Jørgensen;Z. Földes

为了提高对扩散链运动导致蛋白质折叠的理解，我们利用荧光相关光谱（FCS）研究了变性酵母细胞色素c的微秒级构象波动。我们表明，从染料Alexa-488化学连接到蛋白质上发出的荧光取决于链的延伸，这样链长的波动将产生荧光强度的波动。暴露于化学变性剂导致扩散时间增加，表明分子膨胀。未折叠蛋白质的结构波动引起发射荧光的波动。然而，FCS测量未能显示链段的构象波动，在变性状态下链波动的时间尺度上建立了4 µs的上限。这清楚地表明，在折叠实验中观察到的时间常数为50 µs的早期过程必须涉及自由能垒的通过，而不是已经提出的无垒链崩溃。

In order to improve the understanding of the diffusive chain movements leading to protein folding, we have studied microsecond conformational fluctuations in denatured yeast cytochrome c by fluorescence correlation spectroscopy (FCS). We show that emitted fluorescence from the dye Alexa-488 chemically attached to the protein depends on the extension of the chain, such that fluctuations in chain length will give fluctuations in fluorescence intensity. Exposure to chemical denaturants leads to an increase in diffusion times, indicating expansion of the molecule. Structural fluctuations of the unfolded protein give rise to fluctuations in the emitted fluorescence. However, FCS measurements fail to show conformational fluctuations of chain segments, establishing an upper bound of 4 µs on the timescale of chain fluctuations in the denatured state. This clearly shows that an early process with a time constant of 50 µs observed in folding experiments must involve passage of a free energy barrier, and cannot be barrierless chain collapse as has been proposed.