Automated Pre-screening System for in meso Membrane protein crystallization

描述(申请人提供)：在脂类中间相中结晶膜蛋白是一种很有前途的技术，最近产生了高分辨率的人？2-肾上腺素能和腺苷A2A2AG蛋白偶联受体结构。在介观结晶中的一个显著特征是，目标蛋白被嵌入到高度弯曲和折叠的空间脂双层中，形成脂类立方相。立方相的结构对大蛋白质或低聚蛋白质聚集体的扩散施加了空间限制。我们的初步数据表明，中观结晶实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。这种事件没有视觉反馈，立方相仍然清晰，因为非扩散粘着的蛋白质寡聚聚集体的尺寸远低于100 nm。蛋白质的聚集行为取决于特定的蛋白质结构、宿主脂类和用于结晶的添加剂。我们建议开发一种自动化系统，用于对MESO中的膜蛋白进行光漂白后荧光恢复(FRAP)研究。该系统将用于建立一种预筛选方法，该方法将使用不到10 L的荧光标记蛋白质溶液来筛选蛋白质在与精心挑选的普通沉淀剂的96种溶液孵育的脂质立方相中的扩散。本实验的结果将有助于选择合适的介观结晶蛋白结构，并消除后续结晶试验中的某些沉淀物。这种结晶预筛选方法将显着增加获得初始晶体命中的机会，从而确定具有挑战性的膜蛋白的更高分辨率结构，这对于设计副作用较小的新型和改进的高特异性药物至关重要。 公共卫生建议(申请人提供)：该研究项目将开发新技术，以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点。结晶将促进蛋白质三维结构的溶解，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.