Sazaki Gen, Van Driessche Alexander E S, Dai Guoliang, Okada Masashi, Matsui Takuro, Otálora Fermin, Tsukamoto Katsuo, Nakajima Kazuo
Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan.
Protein Pept Lett. 2012 Jul;19(7):743-60. doi: 10.2174/092986612800793118.
To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.
为了开始系统地研究蛋白质晶体的质量改进，必须首先全面阐明蛋白质晶体的基本生长过程。原子力显微镜（AFM）在阐明蛋白质晶体的基本生长过程方面做出了巨大贡献，并证实蛋白质晶体与无机和低分子量化合物晶体一样，利用台阶上的扭折逐层生长。然而，AFM悬臂的扫描极大地扰乱了生长中的蛋白质晶体附近的浓度分布和溶液流动。AFM也无法可视化蛋白质晶体表面上可移动溶质和杂质分子的动态行为。为了弥补AFM的这些缺点，最近已经通过两种先进的光学显微镜进行了原位观察。为了无创地观察蛋白质晶体的基本台阶，开发了结合微分干涉对比显微镜的激光共聚焦显微镜（LCM-DIM）。为了可视化单个可移动蛋白质分子，将在生物物理学领域广泛使用的全内反射荧光（TIRF）显微镜应用于蛋白质晶体表面的可视化。在这篇综述中，概述了在蛋白质晶体表面基本台阶和单个可移动蛋白质分子的无创原位观察方面的最新进展。
