Department of Physics, University of Northern Iowa, Cedar Falls, IA 50614, USA.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, CO 80045, USA.
Nucleic Acids Res. 2021 May 21;49(9):e53. doi: 10.1093/nar/gkab087.
Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA's utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.
细胞内的分子机器动态地组装、拆卸和重组。可以在单分子水平上观察到它们组件之间的分子相互作用，并使用共定位单分子光谱法进行定量，在该方法中，单个标记分子被短暂地观察到与表面固定的伴侣结合，或者使用其他全内反射荧光显微镜方法，其中相互作用导致标记表面固定伴侣的荧光发生变化。当多个相互作用的伴侣可以形成三元、四元或更高阶复合物时，可以根据复合物的组成部分的出现和重组的顺序来推断这些复合物的空间和时间组织类型。通过在多个通道中荧光行为的变化可以跟踪复合物结构的时间演化。在这里，我们描述了动力学事件解析算法（KERA），这是一种用于组织和分类来自一系列单分子实验的离散荧光轨迹的软件工具。KERA 通过过渡模式将数据分组，并为每个相互作用序列显示详尽的停留时间数据。枚举和量化分子相互作用的序列提供了有关组装、大分子复合物的动力学和结构的潜在机制的重要信息。我们通过分析两种 DNA 结合蛋白（复制蛋白 A 和着色性干皮病互补组 D 解旋酶）的构象动力学来证明 KERA 的实用性。
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