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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Development of a technique for the investigation of folding dynamics of single proteins for extended time periods
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Development of a technique for the investigation of folding dynamics of single proteins for extended time periods

机译：研究延长单个蛋白质折叠动力学的技术

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摘要

A technique was developed for the detection of fluorescence signals from free single molecules for extended time periods and was applied to the characterization of the unfolded states of iso-1-cytochrome c (cyt c). Protein molecules labeled with fluorescent dye were slowly injected into a capillary at concentrations that allow for the observation of one molecule at a time. A laser was introduced into the capillary coaxially, and the fluorescence was imaged as traces by using a lens with a large focal depth and wide field of view. Thus, the traces reflect the time-dependent changes in the fluorescence signals from single proteins. Cyt c was labeled with Alexa Fluor 532 at the C-terminal cysteine (cyt c-Alexa). In bulk experiments, cyt c-Alexa was shown to possess different fluorescence intensity for the native state, the unfolded state (U), and the intermediate state. Single-molecule traces of cyt c-Alexa were recorded by using the device. Intensity histograms of the traces revealed two distributions with broad and narrow widths, which were interpreted to correspond to the U and intermediate state, respectively, observed in the bulk measurements. The broad width of the U suggested the existence of a relatively slow conformational dynamics, which might be consistent with the correlation time (≈15 ms) estimated from the traces assignable to the U. The technique was expected to reveal dynamics of proteins along the folding processes without artifacts caused by immobilization.
机译：开发了一种技术，用于检测来自游离单分子的荧光信号的时间延长，并将其用于表征异-1-细胞色素c（cyt c）的未折叠状态。将标记有荧光染料的蛋白分子缓慢注入毛细管中，其浓度应允许一次观察一个分子。将激光同轴地引入毛细管中，并且通过使用具有大焦深和宽视场的透镜将荧光成像为迹线。因此，迹线反映了来自单个蛋白质的荧光信号随时间的变化。 Cyt c在C端半胱氨酸（cyt c-Alexa）处被Alexa Fluor 532标记。在大量实验中，表明cyt c-Alexa对天然状态，未折叠状态（U）和中间状态具有不同的荧光强度。使用该设备记录了cyt c-Alexa的单分子痕迹。迹线的强度直方图显示出宽和窄宽度的两个分布，这被解释为分别对应于在批量测量中观察到的U和中间状态。 U的宽宽度表明存在相对较慢的构象动力学，这可能与根据可分配给U的迹线估计的相关时间（≈15ms）一致。该技术有望揭示折叠过程中蛋白质的动力学过程中没有因固定而造成的伪影。

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