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A Method for Extracting theFree Energy Surface andConformational Dynamics of Fast-Folding Proteins from Single MoleculePhoton Trajectories

机译：一种提取方法自由能表面和单分子快速折叠蛋白的构象动力学光子轨迹

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Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-stateto one-state downhill folding, as a function of relevant experimentalvariables such as photon count rate, amount of input data, and backgroundnoise. The tests demonstrate that the analysis can accurately retrievethe original one-dimensional free energy surface and microsecond foldingdynamics in spite of the sub-megahertz photon count rates and significantbackground noise levels of current single molecule fluorescence experiments.Therefore, our approach provides a powerful tool for the quantitativeanalysis of single molecule FRET experiments of fast protein foldingthat is also potentially extensible to the analysis of any other biomolecularprocess governed by sub-millisecond conformational dynamics.
机译：单分子荧光光谱法有望提供对蛋白质折叠自由能态和构象运动的直接测量。然而，由于技术限制，尤其是难于分析通常从单个生物分子记录的每毫秒小光子包的技术限制，无法实现这一诺言。这种限制削弱了准确确定构象分布和解析亚毫秒级过程的能力。在这里，我们开发了一种分析程序，可直接从单分子FRET实验产生的带时间戳的光子到达轨迹中提取快速折叠蛋白的构象分布和动力学。我们的程序将Gopich和Szabo最初开发的最大似然分析与描述蛋白质折叠作为一维自由能表面上的扩散的统计力学模型相结合。使用随机动力学模拟，我们彻底测试了该方法在识别各种快速折叠场景中的性能，包括两种状态到一个状态的下坡折叠，根据相关实验变量，例如光子计数率，输入数据量和背景噪声。测试表明，该分析可以准确检索原始的一维自由能表面和微秒级折叠亚兆赫级光子计数率和显着性的动力学当前单分子荧光实验的背景噪声水平。因此，我们的方法为定量分析提供了强大的工具蛋白质快速折叠的单分子FRET实验分析这也可能扩展到任何其他生物分子的分析亚毫秒构象动力学控制着这一过程。

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