Automated Pre-screening System for in meso Membrane protein crystallization

描述（由申请人提供）：脂质中间期（in meso）膜蛋白的结晶是一项很有前途的技术，最近获得了人类？2-肾上腺素能和腺苷A2A G蛋白偶联受体。中介观结晶的一个显著特征是目标蛋白被嵌入高度弯曲折叠的空间脂质双分子层中，形成脂质立方相。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限制。我们的初步数据表明，中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。对于这种事件没有视觉反馈，立方相仍然清晰，因为非扩散粘滞的蛋白质寡聚物聚集体的大小远低于100纳米。蛋白质的聚集行为取决于特定的蛋白质结构、宿主脂质和用于结晶的添加剂。我们建议开发一种自动化系统，用于光漂白后膜蛋白的荧光恢复（FRAP）研究。该系统将用于开发一种预筛选试验，该试验将使用少于10 ？L的荧光标记蛋白溶液，以筛选蛋白质在脂质立方相中的扩散，与96溶液精心选择的常见沉淀剂孵卵。本试验的结果将有助于选择合适的中介晶化蛋白结构，并在随后的结晶试验中消除某些沉淀剂。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，从而确定更具挑战性的膜蛋白的高分辨率结构，这对于设计新的和改进的高特异性药物具有更小的副作用至关重要。

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.