Studies on defect generation mechanisms in protein crystals by molecular-level in situ observations of elementary growth processes

利用四方模型蛋白质溶菌酶晶体，利用激光共聚焦显微镜结合差示干涉显微镜(LCM-DIM)、单分子可视化技术(SMV)和激光光散射层析(LLST)等先进光学显微镜，原位观察到(I)晶体表面的基本生长步骤，(II)单个蛋白质分子在晶体-溶液界面上的扩散和(III)晶体内部的缺陷。本研究的主要结果如下：1)流动的影响：用LCM-DIM对强迫流动下晶体表面生长台阶的原位观察使我们能够发现流动显著地加速了杂质向晶体表面的传输，并提供了聚集的台阶。2)表面扩散：我们首次成功地用SMV原位观察到单个荧光标记的溶菌酶(F-L)分子在晶体表面附近扩散。我们发现F-L分子在晶体溶胶…上的扩散3)杂质蛋白质的吸附机理：通过原位观察F-L和荧光标记的溶菌酶二聚体(F-D)同时在晶体表面的吸附过程，我们发现F-L优先吸附在台阶上，而F-D随机吸附在平台上。F-L和F-D的不同吸附位置为杂质效应提供了不同的过饱和依赖关系。4)晶体内部缺陷：我们成功地用LCM-DIM原位观察到了生长中的晶体内部的位错和包裹体，揭示了加入到种子晶体中的微晶体产生了许多位错，从而在被结合的微晶体的顶部形成了螺旋生长的山丘。我们还成功地可视化了LST的对比，这些对比可能是由晶体中的空位和杂质粒子引起的微缺陷。较少

Using tetragonal crystals of model protein lysozyme, we observed in situ (i) elementary growth steps on a crystal surface, (ii) individual protein molecules diffusing at a crystal-solution interface and (iii) defects inside a crystal by advanced optical microscopy : laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM), a single molecule visualization technique (SMV) and laser light scattering tomography (LLST). Key results found in this study were as follows.1) Effects of flow : In situ observation of growth steps on a crystal surface under forced flow by LCM-DIM enable us to find that flow significantly accelerates the transport of impurity to a crystal surface, and then provide bunched steps.2) Surface diffusion : We succeeded in the in situ observation of individual fluorescent-labeled lysozyme (F-L) molecules diffusing in the vicinity of a crystal surface by SMV, for the first time. We found that diffusion of F-L molecules at a crystal-sol … More ution interface is 4 orders of magnitude slower than that in a bulk solution, and anisotropy of diffusion exists according to crystallographic directions.3) Adsorption mechanism of impure protein : From in situ observation of adsorption processes of F-L and fluorescent-labeled dimmer of lysozyme (F-D) on a crystal surface by both LCM-DIM and SMV simultaneously, we demonstrated that F-L adsorbs preferentially on a step, whereas F-D adsorbs randomly on a terrace. The different adsorption sites of F-L and F-D provided different supersaturation dependencies of impurity effects.4) Defects inside crystals : We succeeded in observing in situ dislocations and inclusions inside a growing crystal by LCM-DIM, and revealed that microcrystals incorporated in a seed crystal generate many dislocations and hence provide spiral growth hillocks on top of microcrystals incorporated. We also succeeded in visualizing the contrasts that are probably microdefects originated from vacancies and impurity particles inside a crystal by LLST. Less