Kojima Hiroaki, Toba Shiori, Sakakibara Hitoshi, Oiwa Kazuhiro
Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Nishi-ku, Japan.
Methods Cell Biol. 2009;92:83-105. doi: 10.1016/S0091-679X(08)92006-9. Epub 2009 Nov 21.
In recent decades, the development of technologies such as optical trap nanometry and advanced fluorescence microscopy have provided tools for studying the dynamics of single protein molecules in vitro and in vivo with nanometer precision over timescales from milliseconds to seconds. The single-molecule sensitivities of these methods permit studies to be made on conformational changes and dynamics of protein molecules that are masked in ensemble-averaged experiments. For protein motors, force generation, processivity, step size, transitions among mechanical states, and mechanochemical coupling are among the properties that can be directly measured by single-molecule techniques. Our understanding of the functions of protein motors has thus benefited considerably from the application of single-molecule techniques. This chapter will focus on single-molecule techniques applicable to axonemal dyneins, the principles upon which they work and how they are constructed and conducted.
近几十年来，诸如光镊纳米测量技术和先进荧光显微镜等技术的发展，为在体外和体内以纳米精度研究单个蛋白质分子在从毫秒到秒的时间尺度上的动力学提供了工具。这些方法的单分子灵敏度使得能够对在整体平均实验中被掩盖的蛋白质分子的构象变化和动力学进行研究。对于蛋白质马达，力的产生、持续性、步长、机械状态之间的转变以及机械化学偶联等特性都可以通过单分子技术直接测量。因此，我们对蛋白质马达功能的理解从单分子技术的应用中受益匪浅。本章将重点介绍适用于轴丝动力蛋白的单分子技术、它们的工作原理以及构建和实施方式。
