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首页> 外文期刊>Crystal growth & design >Development of an automated high throughput LCP-FRAP assay to guide membrane protein crystallization in lipid mesophases
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Development of an automated high throughput LCP-FRAP assay to guide membrane protein crystallization in lipid mesophases

机译：自动化的高通量LCP-FRAP分析方法的开发，可指导脂质中间相中的膜蛋白结晶

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Crystallization in lipidic mesophases (in meso) has been successfully used to obtain a number of high-resolution membrane protein structures including challenging members of the human G protein-coupled receptor (GPCR) family. Crystallogenesis in arguably the most successful mesophase, lipidic cubic phase (LCP), critically depends on the ability of protein to diffuse in the LCP matrix and to form specific protein-protein contacts to support crystal nucleation and growth. The ability of an integral membrane protein to diffuse in LCP is strongly affected by the protein aggregation state, the structural parameters of LCP, and the chemical environment. In order to satisfy both requirements of diffusion and specific interactions, one must balance multiple parameters, such as identity of LCP host lipid, composition of precipitant solution, identity of ligand, and protein modifications. Screening within such multidimensional crystallization space presents a significant bottleneck in obtaining initial crystal leads. To reduce this combinatorial challenge, we developed a precrystallization screening assay to measure the diffusion characteristics of a protein target in LCP. Utilizing the fluorescence recovery after photobleaching (FRAP) technique in an automated and high throughput manner, we were able to map conditions that support adequate diffusion in LCP using a minimal amount of protein. Data collection and processing protocols were validated using two model GPCR targets: the β_2-adrenergic receptor and the A_(2A) adenosine receptor.
机译：脂质中间相（中间相）的结晶已成功用于获得许多高分辨率的膜蛋白结构，包括人G蛋白偶联受体（GPCR）家族的具有挑战性的成员。可以说，最成功的中间相，脂质立方相（LCP）的晶体形成关键取决于蛋白质在LCP基质中扩散并形成特定的蛋白质-蛋白质接触以支持晶体成核和生长的能力。完整的膜蛋白在LCP中扩散的能力受蛋白聚集状态，LCP的结构参数和化学环境的强烈影响。为了满足扩散和特定相互作用的需求，必须平衡多个参数，例如LCP宿主脂质的身份，沉淀溶液的组成，配体的身份和蛋白质修饰。在这样的多维结晶空间内进行筛查是获得初始晶体引线的重大瓶颈。为了减少这种组合挑战，我们开发了一种预结晶筛选测定法来测量LCP中蛋白质靶标的扩散特征。利用光漂白（FRAP）技术后的荧光回收率，以自动化且高通量的方式，我们能够绘制出支持使用最小量蛋白质在LCP中充分扩散的条件。数据收集和处理协议已验证使用两个模型GPCR目标：β_2-肾上腺素受体和A_（2A）腺苷受体。

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