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

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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