Harris Paul David, Lerner Eitan
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
Biophys Rep (N Y). 2022 Sep 14;2(3). doi: 10.1016/j.bpr.2022.100071. Epub 2022 Aug 17.
Single-molecule spectroscopy has revolutionized molecular biophysics and provided means to probe how structural moieties within biomolecules spatially reorganize at different timescales. There are several single-molecule methodologies that probe local structural dynamics in the vicinity of a single dye-labeled residue, which rely on fluorescence lifetimes as readout. Nevertheless, an analytical framework to quantify dynamics in such single-molecule single dye fluorescence bursts, at timescales of microseconds to milliseconds, has not yet been demonstrated. Here, we suggest an analytical framework for identifying and quantifying within-burst lifetime-based dynamics, such as conformational dynamics recorded in single-molecule photo-isomerization-related fluorescence enhancement. After testing the capabilities of the analysis on simulations, we proceed to exhibit within-burst millisecond local structural dynamics in the unbound -synuclein monomer. The analytical framework provided in this work paves the way for extracting a full picture of the energy landscape for the coordinate probed by fluorescence lifetime-based single-molecule measurements.
单分子光谱学彻底改变了分子生物物理学，并提供了手段来探究生物分子中的结构部分如何在不同时间尺度上进行空间重组。有几种单分子方法可探测单个染料标记残基附近的局部结构动力学，这些方法依赖于荧光寿命作为读出信号。然而，尚未证明有一个分析框架能够在微秒到毫秒的时间尺度上量化此类单分子单染料荧光猝发中的动力学。在此，我们提出了一个分析框架，用于识别和量化基于猝发寿命的动力学，例如在单分子光异构化相关荧光增强中记录的构象动力学。在通过模拟测试了该分析的能力之后，我们接着展示了未结合的α-突触核蛋白单体中的猝发内毫秒级局部结构动力学。这项工作中提供的分析框架为基于荧光寿命的单分子测量所探测的坐标提取能量景观的全貌铺平了道路。
