应用全内反射单分子荧光成像研究蛋白复合物亚基组成

细胞的生化过程大都是由蛋白复合物完成的,研究蛋白复合物亚基的组成对于了解蛋白质的结构和生物学功能具有重要的意义,然而如何准确确定蛋白复合物中蛋白质亚基的数量（stoichiometry）仍然是一个挑战.近年来,活细胞体系单分子荧光成像技术的不断发展为原位实时动态地研究蛋白质的结构和性质提供了新的手段.本文主要介绍了应用活细胞全内反射单分子荧光成像技术表征细胞膜区蛋白复合物组成的3种方法,包括单分子漂白步数分析、荧光强度统计分布以及蛋白运动分析,并结合其基本原理介绍了这几种方法在活细胞体系膜蛋白研究中的应用.

Study of protein stoichiometry by single-molecule imaging with total internal reflection fluorescence microscopy

Most of cellular processes are realized by protein complexes,and the study of subunits composition of protein complexes is very important for the understanding of protein structure and biological function.However,accurate determination of protein stoichiometry under the physiological conditions is still a challenge.In recent years,the development of single-molecule fluorescence imaging of living cells offers a new approach to characterize protein in real time.In this review,we first introduce the basic principle of imaging single-molecule in living cells by total internal reflection fluorescence microscopy（TIRFM） and then discuss the application of single-molecule TIRFM to study the plasma membrane protein complex in three ways,including the analysis of photobleaching step,the statistical distribution of fluorescence intensity and the dynamics of protein diffusion.