Direct observation of adsorption sites of protein impurities and their effects on step advancement of protein crystals
Direct observation of adsorption sites of protein impurities and their effects on step advancement of protein crystals
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DOI：
10.1021/cg8006684
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发表时间：
2009-05
影响因子：
3.8
通讯作者：
Van Driessehe Alexander Es;G. Sazaki;戴国亮;F. Otálora;Gavira Ja;T. Matsui;I. Yoshizaki;K. Tsukamoto;K. Nakajima
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作者：
Van Driessehe Alexander Es;G. Sazaki;戴国亮;F. Otálora;Gavira Ja;T. Matsui;I. Yoshizaki;K. Tsukamoto;K. Nakajima
我们通过激光共聚焦显微镜结合微分干涉对比显微镜，在不同过饱和度下，对四方溶菌酶晶体{110}面上的基本二维（2D）岛的台阶速度进行了无创测量。我们使用三种蛋白质：荧光标记的溶菌酶（F - 溶菌酶）、溶菌酶的共价键二聚体（二聚体）和一种18 kDa的多肽（18 kDa），研究了蛋白质杂质对基本台阶生长的影响及其在晶体表面吸附位点之间的相关性。这三种蛋白质杂质都抑制了台阶的推进。然而，它们对台阶速度抑制的过饱和度依赖性不同。为了阐明这种差异的原因，我们通过单分子可视化原位观察了F - 溶菌酶和荧光标记二聚体（F - 二聚体）单个分子在晶体表面的吸附位点。我们发现F - 溶菌酶优先吸附在台阶（即扭折处），而F - 二聚体随机吸附在平台上。考虑到F - 溶菌酶和F - 二聚体不同的吸附位点，我们能够成功解释杂质对台阶速度的不同影响。这些观察结果有力地表明18 kDa也随机吸附在平台上。生化学溶菌酶表现出一种复杂的效应，这种效应不能仅由生化学溶菌酶中存在的两种主要杂质（二聚体和18 kDa）来解释，这表明微量的其他杂质显著影响台阶的推进。
We measured noninvasively step velocities of elementary two-dimensional (2D) islands on {110} faces of tetragonal lysozyme crystals, under various supersaturations, by laser confocal microscopy combined with differential interference contrast microscopy. We studied the correlation between the effects of protein impurities on the growth of elementary steps and their adsorption sites on a crystal surface, using three kinds of proteins: fluorescent-labeled lysozyme (F-lysozyme), covalently bonded dimers of lysozyme (dimer), and a 18 kDa polypeptide (18 kDa). These three protein impurities suppressed the advancement of the steps. However, they exhibited different supersaturation dependencies of the suppression of the step velocities. To clarify the cause of this difference, we observed in situ the adsorption sites of individual molecules of F-lysozyme and fluorescent-labeled dimer (F-dimer) on the crystal surface by single-molecule visualization. We found that F-lysozyme adsorbed preferentially on steps (i.e., kinks), whereas F-dimer adsorbed randomly on terraces. Taking into account the different adsorption sites of F-lysozyme and F-dimer, we could successfully explain the different effects of the impurities on the step velocities. These observations strongly suggest that 18 kDa also adsorbs randomly on terraces. Seikagaku lysozyme exhibited a complex effect that could not alone be explained by the two major impurities (dimer and 18 kDa) present in Seikagaku lysozyme, indicating that trace amounts of other impurities significantly affect the step advancement.