Howard Hughes Medical Institute , Baltimore , Maryland 21205 , United States.
Chem Rev. 2020 Jan 8;120(1):36-78. doi: 10.1021/acs.chemrev.9b00361. Epub 2019 Oct 29.
Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.
分子马达是多种酶，它们将化学能量转化为机械功，从而执行关键的细胞功能，如 DNA 复制和转录、DNA 超螺旋、细胞内运输和 ATP 合成。单分子技术已被广泛用于鉴定分子马达反应循环中的结构中间体，并了解能量消耗的亚步骤如何驱动中间体之间的转变。在这里，我们综述了广泛的单分子工具和技术，如光学和磁性镊子、原子力显微镜 (AFM)、单分子荧光共振能量转移 (smFRET)、纳米孔镊子和增加可观测数量的混合技术。这些方法通过施加力和扭矩来操纵单个生物分子，并观察单个马达复合物中的动态构象变化。我们还综述了这些技术如何应用于研究各种马达，如解旋酶、DNA 和 RNA 聚合酶、拓扑异构酶、核小体重塑酶以及参与 DNA 凝聚、分离和消化的马达。对分子马达中机械化学耦联的深入分析使得人工工程马达的发展成为可能。我们综述了诸如诱变、化学修饰和光遗传学等技术，这些技术已被用于改造现有分子马达，例如改变速度、进程或功能。我们还讨论了对工程马达的单分子分析如何使我们能够挑战我们对分子马达如何传递能量的基本理解。
