Rhoades Elizabeth, Cohen Mati, Schuler Benjamin, Haran Gilad
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel.
J Am Chem Soc. 2004 Nov 17;126(45):14686-7. doi: 10.1021/ja046209k.
The folding dynamics of small proteins are often described in terms of a simple two-state kinetic model. Within this notion, the behavior of individual molecules is expected to be stochastic, with a protein molecule residing in either the unfolded or the folded state for extended periods of time, with intermittent rapid jumps across the free energy barrier. However, a direct observation of this bistable behavior has not been made to date. Rather, previous reports of folding trajectories of individual proteins have shown an unexpected degree of complexity. This raises the question whether the simple kinetic properties derived from classical experiments on large ensembles of molecules are reflected in the folding paths taken by individual proteins. Here we report single-molecule folding/unfolding trajectories observed by fluorescence resonance energy transfer for a protein that meets all criteria of a two state-system. The trajectories, measured on molecules immobilized in lipid vesicles, demonstrate the anticipated bistable behavior, with steplike transitions between folded and unfolded conformations. They further allow us to put an upper bound on the barrier crossing time.
小蛋白质的折叠动力学通常用一个简单的两态动力学模型来描述。在这个概念中，单个分子的行为预计是随机的，蛋白质分子会长时间处于未折叠或折叠状态，在自由能垒上有间歇性的快速跳跃。然而，迄今为止尚未对这种双稳态行为进行直接观察。相反，先前关于单个蛋白质折叠轨迹的报道显示出了意想不到的复杂程度。这就提出了一个问题，即从对大量分子集合进行的经典实验中得出的简单动力学特性是否反映在单个蛋白质的折叠路径中。在这里，我们报告了通过荧光共振能量转移观察到的一种符合两态系统所有标准的蛋白质的单分子折叠/解折叠轨迹。在固定于脂质囊泡中的分子上测量的这些轨迹展示了预期的双稳态行为，在折叠和未折叠构象之间有阶梯状转变。它们还使我们能够确定穿越能垒时间的上限。
