Yokota Hiroaki, Kaseda Kuniyoshi, Matsuura Hideyuki, Arai Yushiyuki, Iwane Atsuko, Ishii Yoshiharu, Kodama Takao, Yanagida Toshio
Single Molecule Processes Project, ICORP JST, Osaka 562-0035, Japan.
J Nanosci Nanotechnol. 2004 Jul;4(6):616-21. doi: 10.1166/jnn.2004.009.
In recent years, the development of single-molecule detection techniques has allowed the dynamic properties of biomolecules, which are normally obscured in conventional ensemble measurements, to be measured. One of these single-molecule detection techniques allows the measurement of dissociation and association events of individual molecules to be measured. This technique is based on the unique premise that the mobility between molecules that are bound and the mobility between those that are free in solution are different. The binding of ATP at the beginning and its dissociation at the end of the hydrolysis reaction were detected at the single-molecule level in real time. In this study, we extended this technique to image the dynamic interactions between large biomolecules (protein/protein and protein/polysaccharide). The binding and dissociation of fluorescently labeled macromolecules to partner molecules fixed on a glass surface were visualized by total internal reflection fluorescence microscopy. The dynamic interactions between the proteins in two energy conversion systems, that is, signaling proteins and enzyme molecules moving on dextran, have been measured. In these systems, the dynamic interactions were sensitive to the factors determining the chemical reactions. Thus, the dynamic interactions monitored in the single-molecule measurements provided useful information to further the understanding of the underlying mechanisms of energy conversion systems.
近年来，单分子检测技术的发展使得生物分子的动态特性得以测量，而这些特性在传统的整体测量中通常是模糊不清的。其中一种单分子检测技术能够测量单个分子的解离和结合事件。该技术基于一个独特的前提，即结合态分子之间的迁移率与溶液中游离态分子之间的迁移率不同。在水解反应开始时ATP的结合及其结束时的解离在单分子水平上被实时检测到。在本研究中，我们扩展了这项技术，以成像大型生物分子（蛋白质/蛋白质和蛋白质/多糖）之间的动态相互作用。通过全内反射荧光显微镜观察荧光标记的大分子与固定在玻璃表面的伙伴分子的结合和解离。已经测量了两个能量转换系统中蛋白质之间的动态相互作用，即信号蛋白和在葡聚糖上移动的酶分子。在这些系统中，动态相互作用对决定化学反应的因素敏感。因此，在单分子测量中监测到的动态相互作用为进一步理解能量转换系统的潜在机制提供了有用信息。
