Buscaglia Marco, Schuler Benjamin, Lapidus Lisa J, Eaton William A, Hofrichter James
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Bethesda, MD 20892-0520, USA.
J Mol Biol. 2003 Sep 5;332(1):9-12. doi: 10.1016/s0022-2836(03)00891-x.
Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein ( approximately 10(4)s(-1)). Experiments on the unfolded protein without the added C-terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule Förster resonance energy transfer (FRET) experiments.
半胱氨酸对色氨酸三重态的猝灭为测量无序多肽中特定分子内接触形成的速率提供了一种新工具。在此，我们使用该技术来研究嗜热栖热袍菌的一种小型冷休克蛋白CspTm变性状态下的接触形成，该蛋白经工程改造后在C端含有一个色氨酸残基（W29）和一个半胱氨酸残基（C67）。在所有变性剂浓度下，未折叠蛋白的W29三重态的衰减速率比天然蛋白观察到的速率快十多倍（约10⁴ s⁻¹）。对未添加C端半胱氨酸残基的未折叠蛋白进行的实验表明，这种更快的速率完全是由C67的接触猝灭导致的。在有利于折叠的变性剂浓度下，C67在未折叠状态下的猝灭速率增加，这表明未折叠蛋白发生了压缩，正如之前在单分子荧光共振能量转移（FRET）实验中观察到的那样。
