Enhanced crystallization of the Cys18 to Ser mutant of bovine γB crystallin
Enhanced crystallization of the Cys18 to Ser mutant of bovine γB crystallin
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DOI：
10.1006/jmbi.2001.5155
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发表时间：
2001-12-07
影响因子：
5.6
通讯作者：
Benedek, GB
中科院分区：
文献类型：
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作者：
Asherie, N;Pande, J;Benedek, GB
γ晶状体蛋白（眼晶状体蛋白家族）的半胱氨酸残基参与这些蛋白的聚集和相分离。这两种现象都与白内障的形成有关。我们使用牛γβ晶状体蛋白作为模型系统来研究单个半胱氨酸残基在γ晶状体蛋白聚集和相分离中的作用。在这里，我们比较了重组野生型蛋白 (WT) 和 Cys18 to Ser (C18S) 突变体的热力学和动力学行为。我们发现两种蛋白质的溶解度相似。然而，结晶动力学不同。 WT 结晶速度足够慢，可以轻松观察到亚稳态液-液共存。另一方面，C18S 结晶迅速；纯突变体的亚稳态共存液相不形成。然而，如果结晶在动力学上受到抑制，则可以确定 C18S 的共存曲线。这样我们发现共存曲线与WT的共存曲线重合。尽管结晶动力学存在差异，但这两种蛋白质被发现具有相同的晶体形式和几乎相同的 X 射线结构。我们的结果表明，即使是保守的点突变也可以带来结晶动力学的巨大变化。讨论了我们的发现对白内障形成和蛋白质结晶的影响。 (C) 2001 年学术出版社。
The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammabeta crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed. (C) 2001 Academic Press.