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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >From the Cover: Feature Article: Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories
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From the Cover: Feature Article: Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories

机译：从封面开始：专题文章：从单分子光子到光子轨迹的蛋白质折叠中过渡路径时间上限的实验确定

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Transition paths are a uniquely single-molecule property not yet observed for any molecular process in solution. The duration of transition paths is the tiny fraction of the time in an equilibrium single-molecule trajectory when the process actually happens. Here, we report the determination of an upper bound for the transition path time for protein folding from photon-by-photon trajectories. FRET trajectories were measured on single molecules of the dye-labeled, 56-residue 2-state protein GB1, immobilized on a glass surface via a biotin-streptavidin-biotin linkage. Characterization of individual emitted photons by their wavelength, polarization, and absolute and relative time of arrival after picosecond excitation allowed the determination of distributions of FRET efficiencies, donor and acceptor lifetimes, steady state polarizations, and waiting times in the folded and unfolded states. Comparison with the results for freely diffusing molecules showed that immobilization has no detectable effect on the structure or dynamics of the unfolded protein and only a small effect on the folding/unfolding kinetics. Analysis of the photon-by-photon trajectories yields a transition path time <200 μs, >10,000 times shorter than the mean waiting time in the unfolded state (the inverse of the folding rate coefficient). Szabo's theory for diffusive transition paths shows that this upper bound for the transition path time is consistent with previous estimates of the Kramers preexponential factor for the rate coefficient, and predicts that the transition path time is remarkably insensitive to the folding rate, with only a 2-fold difference for rate coefficients that differ by 105-fold.
机译：过渡路径是溶液中任何分子过程都尚未观察到的独特的单分子性质。当过程实际发生时，过渡路径的持续时间是平衡单分子轨迹中时间的一小部分。在这里，我们报告确定从光子到光子轨迹的蛋白质折叠的过渡路径时间的上限。在通过生物素-链霉亲和素-生物素键合固定在玻璃表面上的染料标记的，具有56个残基的2状态蛋白质GB1的单分子上测量FRET轨迹。通过波长，极化以及皮秒激发后到达的绝对和相对到达时间来表征各个发出的光子，可以确定FRET效率的分布，施主和受主的寿命，稳态极化以及在折叠和展开状态下的等待时间。与自由扩散分子的结果比较表明，固定化对未折叠蛋白的结构或动力学没有可检测的影响，而对折叠/展开动力学只有很小的影响。分析逐个光子的轨迹会产生一个过渡路径时间<200μs，比未折叠状态下的平均等待时间短10,000倍（折叠速率系数的倒数）。 Szabo的扩散过渡路径理论表明，该过渡路径时间的上限与速率系数的Kramers指数前因数的先前估计一致，并预测过渡路径时间对折叠率非常不敏感，只有2速率系数的10倍差异，相差10 5 倍。

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