Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories
Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories
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DOI：
10.1073/pnas.0901178106
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发表时间：
2009-07-21
影响因子：
11.1
通讯作者：
Eaton, William A.
中科院分区：
文献类型：
--
作者：
Chung, Hoi Sung;Louis, John M.;Eaton, William A.
跃迁路径是一种独特的单分子性质，尚未在溶液中的任何分子过程中观察到。跃迁路径的持续时间是当过程实际发生时，在平衡单分子轨道上的时间的微小部分。在这里，我们报告的过渡路径时间从光子的光子轨迹的蛋白质折叠的上限的确定。在染料标记的56个残基的2态蛋白GB 1的单分子上测量FRET轨迹，所述蛋白GB 1通过生物素-链霉亲和素-生物素连接固定在玻璃表面上。个别发射的光子的波长，偏振，绝对和相对时间的皮秒激发后到达的表征允许FRET效率，供体和受体的寿命，稳态偏振，并在折叠和未折叠状态的等待时间的分布的测定。与自由扩散分子的结果比较表明，固定化没有可检测到的影响的结构或动力学的展开的蛋白质，只有一个小的影响折叠/展开动力学。逐光子轨迹的分析产生的过渡路径时间比展开状态下的平均等待时间短10，000倍（折叠速率系数的倒数）。Szabo的扩散跃迁路径理论表明，跃迁路径时间的上限与之前对速率系数的Kramers指前因子的估计一致，并预测跃迁路径时间对折叠速率非常不敏感，对于相差10（5）倍的速率系数，只有2倍的差异。
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 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 10(5)-fold.