Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, 81377 München, Germany.
Proc Natl Acad Sci U S A. 2023 Jan 24;120(4):e2211896120. doi: 10.1073/pnas.2211896120. Epub 2023 Jan 18.
Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.
荧光相关光谱学是一种研究生物分子快速构象变化的通用工具，特别是当与Förster 共振能量转移（FRET）结合使用时。尽管有许多方法可用于识别 FRET 实验中的结构动力学，但当涉及两个强度水平时，确定正向和反向跃迁速率常数，从而也确定平衡常数是困难的。在这里，我们通过仅基于其相对于激发脉冲的到达时间（微时间）将子集的光子包含在自相关分析中，将强度相关分析与荧光寿命信息结合起来。通过拟合相关幅度作为微时间门函数，可以获得来自两个荧光强度水平系统的跃迁速率常数和相应的平衡常数。使用模拟和基于 DNA 折纸的模型系统在固定化和自由扩散分子的实验中证明了这种收缩门荧光相关光谱学（sg-FCS）方法。我们进一步表明，sg-FCS 即使涉及暗猝灭剂（在这种情况下为石墨烯）也可以区分光物理和动态强度变化。最后，我们揭示了基于 FRET 的膜电荷传感器的机制，表明该方法具有广泛的潜力。通过 sg-FCS，我们提出了一种不需要先验知识的算法，因此当对时间相关的单光子数据进行自相关分析时，很容易实现。
