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首页> 外文期刊>Journal of Molecular Biology >Rapid collapse precedes the fast two-state folding of the cold shock protein.
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Rapid collapse precedes the fast two-state folding of the cold shock protein.

机译：快速崩溃先于冷休克蛋白的快速两态折叠。

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摘要

The cold shock protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-protein distances during folding by Forster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R0 value of about 22 A was well suited for following distance changes during the folding of this protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor-acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that > or =50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.
机译：冷休克蛋白Bc-Csp在反应中迅速折叠，该反应通过没有中间体的动力学二态机制很好地描述了。我们通过结合Forster共振能量转移（FRET）和停流实验测量了折叠过程中六个蛋白内距离的缩短。将单个色氨酸残基作为供体工程化到蛋白质中，并将单个5-（（（（乙酰氨基）乙基）氨基）萘-1-磺酸盐（AEDANS）残基作为受体放置在Bc-Csp的溶剂暴露位点。它们的R0值约为22 A，非常适合在此蛋白质折叠过程中以高灵敏度跟随距离变化。诱变和标记不会改变重折叠动力学。折叠过程中能量传递的变化通过供体和受体的发射进行了监测，并发现了相互的影响。在两种情况下，在展开和折叠状态下，供体-受体的距离相似，因此，折叠时能量转移的动力学变化很小。对于四个供体/受体对，我们发现折叠时能量转移增加的≥50％发生在折叠的限速步骤之前。这表明折叠时分子内距离的缩短的大约一半已经发生在限速步骤之前，并且表明Bc-Csp的快速二态折叠反应之前是非常迅速的塌陷。

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