We describe theory, experiments, and analyses of three-color Förster resonance energy transfer (FRET) spectroscopy for probing sub-millisecond conformational dynamics of protein folding and binding of disordered proteins. We devise a scheme that uses single continuous-wave laser excitation of the donor instead of alternating excitation of the donor and one of the acceptors. This scheme alleviates photophysical problems of acceptors such as rapid photobleaching, which is crucial for high time resolution experiments with elevated illumination intensity. Our method exploits the molecular species with one of the acceptors absent or photobleached, from which two-color FRET data is collected in the same experiment. We show that three FRET efficiencies and kinetic parameters can be determined without alternating excitation from a global maximum likelihood analysis of two-color and three-color photon trajectories. We implement co-parallelization of CPU-GPU processing, which leads to a significant reduction of the likelihood calculation time for efficient parameter determination.

我们描述了三色Förster共振能量转移（FRET）光谱的理论，实验和分析，用于探测亚毫秒级构象动力学的蛋白质折叠和无序蛋白质的结合。我们设计了一种方案，该方案使用供体的单个连续波激光激发，而不是供体和一个受体的交替激发。该方案减轻了受体的光物理问题，例如快速的光漂白，这对于提高光照强度的高分辨实验至关重要。我们的方法利用了一种不存在或被光漂白的受体分子，在同一实验中从中收集了两种颜色的FRET数据。我们表明，无需对两色和三色光子轨迹的全局最大似然分析进行交替激发，即可确定三个FRET效率和动力学参数。我们实现了CPU-GPU处理的并行并行处理，从而显着减少了有效参数确定的似然计算时间。