Effects of impurities on membrane-protein crystallization in different systems.
Effects of impurities on membrane-protein crystallization in different systems.
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DOI：
10.1107/s0907444909029163
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发表时间：
2009-10
期刊：
影响因子：
--
通讯作者：
Nollert P
中科院分区：
文献类型：
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作者：
Kors CA;Wallace E;Davies DR;Li L;Laible PD;Nollert P
研究了常见杂质对各种膜蛋白结晶体系的影响，发现脂质立方相结晶方法是最稳健的，可耐受高达 50% 的蛋白质污染水平，对晶体质量影响很小。如果普遍适用，可以利用这种耐受性（i）在初始结晶试验中确定给定膜蛋白的“结晶性”和（ii）对部分纯的膜蛋白样品进行结晶试验。当开始蛋白质结晶项目时，科学家们面临着一些未知因素。其中包括以下问题：（i）起始材料的纯度是否足够？ (ii) 哪种类型的结晶实验最有希望进行？纯化用于结晶试验的活性膜蛋白样品的困难以及与生产此类样品相关的高成本需要极其务实的方法。此外，需要实用指南来提高膜蛋白结晶的效率。为了解决这些难题，我们研究了常见杂质对各种膜蛋白结晶体系的影响，发现基于脂质立方相 (LCP) 的结晶方法比洗涤剂环境中使用蒸汽扩散或微批量方法的结晶更稳健，能够耐受蛋白质、脂质或其他一般膜成分形式的污染。基于 LCP 的结晶从含有大量残留杂质的样品中产生了球形红杆菌光合反应中心 (RC) 的晶体。获得的晶体的蛋白质污染水平高达 50%，并且向 RC 纯样品中添加脂质材料和膜碎片对基于 LCP 的结晶筛选中获得的晶体的数量或质量几乎没有影响。如果普遍适用，这种对杂质的耐受性可以避免在进行初始结晶筛选试验以确定可以针对给定目标蛋白优化的初步结晶条件时对超高纯度样品的需要。
The effects of commonly encountered impurities on various membrane-protein crystallization regimes are investigated and it is found that the lipidic cubic phase crystallization methodology is the most robust, tolerating protein contamination levels of up to 50%, with little effect on crystal quality. If generally applicable, this tolerance may be exploited (i) in initial crystallization trials to determine the ‘crystallizability’ of a given membrane-protein and (ii) to subject partially pure membrane-protein samples to crystallization trials. When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein.