LCP-FRAP Assay for Pre-Screening Membrane Proteins for In Meso Crystallization
LCP-FRAP Assay for Pre-Screening Membrane Proteins for In Meso Crystallization
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DOI：
10.1021/cg800778j
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发表时间：
2008-12-01
影响因子：
3.8
通讯作者：
Stevens, Raymond C.
中科院分区：
文献类型：
--
作者：
Cherezov, Vadim;Liu, Jeffrey;Stevens, Raymond C.
用光漂白后的荧光恢复法研究了细菌视紫红质和β2肾上腺素能受体两种完整膜蛋白在脂质立方相(LCP)中的扩散。我们发现，LCP基质中的扩散特性强烈依赖于蛋白质的结构和应用的筛选条件。常见的沉淀剂往往会限制蛋白质在LCP中的扩散，从而阻碍它们结晶的机会。较高的蛋白质流动分数和较快的扩散速度与已知的结晶条件有很好的相关性。利用这一知识，人们现在可以用微克量的材料预先筛选沉淀条件，以排除不利于扩散、成核和晶体生长的条件。本实验的结果将通过确定合适的蛋白质结构、稳定化合物和有利于介观结晶的沉淀条件来缩小膜蛋白的结晶空间。结晶预筛选将显著增加获得初始晶体命中的机会，加速产生具有挑战性的膜蛋白靶标的高分辨率结构的努力。
Fluorescence recovery after photobleaching was used to study the diffusion of two integral membrane proteins, bacteriorhodopsin and beta2-adrenergic receptor, in lipidic cubic phase (LCP). We found that the diffusion properties within the LCP matrix strongly depend on the protein construct and applied screening conditions. Common precipitants often induce restriction on diffusion of proteins in LCP and thereby impede their chances for crystallization. A high protein mobile fraction and a fast diffusion rate correlate well with known crystallization conditions. Using this knowledge, one can now prescreen precipitant conditions with microgram quantities of material to rule out conditions that are not conducive to diffusion, nucleation, and crystal growth. The results of this assay will narrow membrane protein crystallization space by identifying suitable protein constructs, stabilizing compounds and precipitant conditions amenable to in meso crystallization. Crystallization prescreening will significantly increase the chances of obtaining initial crystal hits, expediting efforts in generating high-resolution structures of challenging membrane protein targets.