Automated Pre-screening System for in meso Membrane protein crystallization

描述（由申请人提供）：结晶的膜蛋白在介晶中间相（中）是一种很有前途的技术，最近产生了高分辨率的结构，人类？2-肾上腺素能和腺苷A2A G蛋白偶联受体。介观结晶的一个显著特征是目的蛋白嵌入高度弯曲和折叠的空间脂质双层中，形成无规立方相。立方相的结构对大蛋白质或寡聚蛋白质聚集体的扩散施加空间约束。我们的初步数据表明，介观结晶试验失败的主要原因之一是由于快速的非特异性蛋白质聚集。对这种事件没有视觉反馈，立方相保持透明，因为非扩散的粘附蛋白质寡聚体的大小远低于100 nm。蛋白质的聚集行为取决于特定的蛋白质构建体、宿主脂质和用于结晶的添加剂。我们建议开发一个自动化系统进行光漂白后的荧光恢复（FRAP）的研究中膜蛋白。该系统将被用来开发一个预筛选试验，将使用不到10？L荧光标记的蛋白质溶液，以筛选与96种精心选择的常见沉淀剂溶液孵育的无水立方相中蛋白质的扩散。该测定的结果将有助于选择适合于内消旋结晶的蛋白质构建体，并从随后的结晶试验中消除某些沉淀物。这种结晶预筛选方法将显著增加获得初始晶体命中的机会，导致确定挑战性膜蛋白的更高分辨率结构，这对于设计具有更小副作用的新的和改进的高度特异性药物至关重要。 公共卫生解放（申请人提供）：本研究项目将开发新技术，加快膜蛋白结晶的成功率。这些蛋白质参与重要的细胞和生理过程，因此是重要的药物靶标。结晶将促进蛋白质的三维结构的解决方案，为合理的药物开发过程提供必要的模板。

DESCRIPTION (provided by applicant): Crystallization of membrane proteins in lipidic mesophases (in meso) is a promising technique, which recently yielded high-resolution structures of human ?2-adrenergic and adenosine A2A G protein-coupled receptors. A distinct feature of the in meso crystallization is that the protein of interest is embedded into a highly curved and folded in space lipid bilayer, forming a lipidic cubic phase. The structure of the cubic phase imposes spatial constrains on diffusion of large proteins or oligomeric protein aggregates. Our preliminary data indicate that one of the primary reasons for failure of the in meso crystallization trials is due to a fast nonspecific protein aggregation. There is no visual feedback to such event, the cubic phase remains clear, because the size of the non-diffusing stuck protein oligomeric aggregates is well below 100 nm. The aggregation behavior of a protein depends on the particular protein construct, host lipid and additives employed for crystallization. We propose to develop an automated system for carrying out Fluorescence Recovery after Photobleaching (FRAP) studies of membrane proteins in meso. This system will be used to develop a pre-screening assay which will use less than 10 ?L of fluorescently labeled protein solution to screen for diffusion of the protein in the lipidic cubic phase incubated with 96 solutions of carefully selected common precipitants. The results of this assay will help to select suitable for in meso crystallization protein constructs and to eliminate certain precipitants from subsequent crystallization trials. Such crystallization pre-screening approach will significantly increase chances of obtaining initial crystal hit leading to the determination of more high-resolution structures of challenging membrane proteins essential in designing new and improved highly specific drugs with lesser side effects. PUBLIC HEALTH RELEVENCE (provided by the applicant): This research project will develop new technologies that will accelerate the success rate of membrane protein crystallization. These proteins are involved in important cellular and physiological processes and therefore are important drug targets. Crystallization will facilitate solution of three-dimensional structures of the proteins providing necessary templates for the rational drug development process.