Felekyan Suren, Sanabria Hugo, Kalinin Stanislav, Kühnemuth Ralf, Seidel Claus A M
Institut für Physikalische Chemie, Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Methods Enzymol. 2013;519:39-85. doi: 10.1016/B978-0-12-405539-1.00002-6.
Fluorescence correlation spectroscopy (FCS) in combination with Förster resonance energy transfer (FRET) has been developed to a powerful statistical tool, which allows for the analysis of FRET fluctuations in the huge time of nanoseconds to seconds. FRET-FCS utilizes the strong distance dependence of the FRET efficiency on the donor (D)-acceptor (A) distance so that it developed to a perfect method for studying structural fluctuation in biomolecules involved in conformational flexibility, structural dynamics, complex formation, folding, and catalysis. Structural fluctuations thereby result in anticorrelated donor and acceptor signals, which are analyzed by FRET-FCS in order to characterize underlying structural dynamics. Simulated and experimental examples are discussed. First, we review experimental implementations of FRET-FCS and present theory for a two-state interconverting system. Additionally, we consider a very common case of FRET dynamics in the presence of donor-only labeled species. We demonstrate that the mean relaxation time for the structural dynamics can be easily obtained in most of cases, whereas extracting meaningful information from correlation amplitudes can be challenging. We present a strategy to avoid a fit with an underdetermined model function by restraining the D and A brightnesses of the at least one involved state, so that both FRET efficiencies and both rate constants (i.e., the equilibrium constant) can be determined. For samples containing several fluorescent species, the use of pulsed polarized excitation with multiparameter fluorescence detection allows for filtered FCS (fFCS), where species-specific correlation functions can be obtained, which can be directly interpreted. The species selection is achieved by filtering using fluorescence decays of individual species. Analytical functions for species auto- and cross-correlation functions are given. Moreover, fFCS is less affected by photophysical artifacts and often offers higher contrast, which effectively increases its time resolution and significantly enhances its capability to resolve multistate kinetics. fFCS can also differentiate between species even when their brightnesses are the same and thus opens up new possibilities to characterize complex dynamics. Alternative fluctuation algorithms to study FRET dynamics are also briefly reviewed.
荧光相关光谱法（FCS）与Förster共振能量转移（FRET）相结合已发展成为一种强大的统计工具，它能够分析纳秒到秒的巨大时间范围内的FRET波动。FRET - FCS利用FRET效率对供体（D） - 受体（A）距离的强烈距离依赖性，从而发展成为研究涉及构象灵活性、结构动力学、复合物形成、折叠和催化的生物分子结构波动的理想方法。结构波动会导致供体和受体信号的反相关，FRET - FCS通过分析这些信号来表征潜在的结构动力学。文中讨论了模拟和实验示例。首先，我们回顾了FRET - FCS的实验实现方式，并给出了双态相互转换系统的理论。此外，我们考虑了仅存在供体标记物种时FRET动力学的一种非常常见的情况。我们证明，在大多数情况下，可以轻松获得结构动力学的平均弛豫时间，而从相关幅度中提取有意义的信息可能具有挑战性。我们提出了一种策略，通过限制至少一个相关状态的D和A亮度来避免使用欠定模型函数进行拟合，从而可以确定FRET效率和两个速率常数（即平衡常数）。对于包含多种荧光物种的样品，使用具有多参数荧光检测的脉冲偏振激发可实现滤波FCS（fFCS），由此可以获得物种特异性相关函数，并且可以直接进行解释。通过使用单个物种的荧光衰减进行滤波来实现物种选择。给出了物种自相关和交叉相关函数的解析函数。此外，fFCS受光物理伪像的影响较小，通常具有更高的对比度，这有效地提高了其时间分辨率，并显著增强了其解析多态动力学的能力。即使物种的亮度相同，fFCS也能够区分不同物种，从而为表征复杂动力学开辟了新的可能性。文中还简要回顾了研究FRET动力学的替代波动算法。
