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

通过分子水平原位观察基本生长过程研究蛋白质晶体缺陷产生机制

• 批准号: 16360001
• 负责人: SAZAKI Gen
• 金额: $ 9.86万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16360001/
• 关键词: Protein; Crystal growth; Lattice defects; In situ observation; Elementary growth steps; Surface diffusion; Laser confocal microscopy; Single molecule visualization; レーザー共焦点顕微鏡

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

使用模型蛋白质溶菌酶的四元晶体，我们通过先进的光学显微镜原位观察到（i）晶体表面上的基本生长步骤，（ii）在晶体-溶液界面处扩散的单个蛋白质分子和（iii）晶体内部的缺陷：激光共聚焦显微镜结合微分干涉对比显微镜（LCM-DIM），单分子可视化技术（SMV）和激光散射断层扫描（LLST）。本研究的主要结果如下：1）流动的影响：通过LCM-DIM对强制流动下晶体表面生长台阶的原位观察，我们发现流动显著加速了杂质向晶体表面的传输，从而提供了聚束台阶。2）表面扩散：我们成功地在原位观察单个荧光标记的溶菌酶（F-L）分子扩散在附近的晶体表面的SMV，为第一次。我们发现F-L分子在晶溶胶中的扩散 ...更多信息 扩散界面比本体溶液中慢4个数量级，且扩散存在晶体学方向的各向异性。3）不纯蛋白质的吸附机理：同时用LCM-DIM和SMV原位观察F-L和荧光标记的溶菌酶二聚体（F-D）在晶体表面的吸附过程，发现F-L优先吸附在一个台阶上，而F-D在平台上随机吸附。F-L和F-D的不同吸附位置提供了不同的杂质效应的过饱和依赖性。4）晶体内部的缺陷：我们成功地通过LCM-DIM观察到生长晶体内部的原位位错和夹杂物，并揭示了掺入籽晶中的微晶产生许多位错，因此在掺入的微晶顶部提供螺旋生长小丘。我们还成功地可视化对比度，可能是源于晶体内部的空位和杂质颗粒的微缺陷。少

项目成果

タンパク質の結晶化 : 第11章2節 磁場中で結晶をつくる

蛋白质结晶：第 11 章第 2 节在磁场中创建晶体

• 发表时间: 2005
• 作者: P.Dold;P.Dold;鈴木良尚;鈴木良尚ら;佐崎 元
• 通讯作者: 佐崎 元

Step velocity in tetragonal lysozyme growth as a function of impurity concentration and mass transport conditions

• DOI: 10.1016/j.jcrysgro.2006.04.116
• 发表时间: 2006-07-15
• 期刊: JOURNAL OF CRYSTAL GROWTH
• 影响因子: 1.8
• 作者: Dold, P.;Ono, E.;Sazaki, G.
• 通讯作者: Sazaki, G.

The Use of a new ad hoc growth cell with parallel electrodes for the nucleaion control of lysozyme

使用具有平行电极的新型特设生长池来控制溶菌酶的成核

• 发表时间: 2004
• 期刊: J.Crystal Growth 264
• 作者: A.Moreno;et al.
• 通讯作者: et al.

光学顕微鏡と光学的観察方法

光学显微镜及光学观察方法

• 发表时间: 2005

A novel approach to protein crystallization : Use of a magnetic field and high pressure

蛋白质结晶的新方法：使用磁场和高压

• 发表时间: 2004
• 期刊: Seibutsu Butsuri 44
• 作者: Suzuki;et al.;T.Asai;G.Sazaki
• 通讯作者: G.Sazaki