Core facility infrastructure for optimizing conditions for lipidic cubic phase-based membrane protein crystallization

膜蛋白具有多种生物学功能，对细菌、植物和真核生物的生存至关重要。这些膜蛋白的晶体结构使人们对转运蛋白、信号转导受体和酶的功能有了巨大的了解。然而，膜蛋白的结晶仍然是结构确定的主要瓶颈。利用脂类中间相，如脂立方相(LCP)，已经在获得高分辨晶体结构方面取得了巨大的成功。LCP中膜蛋白结晶成功的主要因素之一是蛋白质在脂双层内扩散的能力。蛋白质的扩散速率受蛋白质聚集、LCP组成和环境的影响，可以通过光漂白后的荧光恢复(FRAP)来测量。FRAP能够测量标记蛋白质的荧光强度在光漂白后在LCP滴内恢复所需的时间量。FRAP允许立即筛选结晶条件，而不必等待数周来确定结晶条件是否适合晶体生长。Formulatrix FRAP是一台独立的成像仪，能够在50分钟内对96孔板进行高通量FRAP漂白和成像。该系统将允许快速筛选LCP脂质成分、沉淀剂成分、配体和蛋白质修饰。Formulatrix FRAP系统在加拿大任何地方都不存在，将成为多伦多整个结构生物学社区的重要资源。此外，Formulatrix FRAP将扩展由教授指导的集成的、多部门的多伦多膜蛋白结晶设施的现有能力。杰弗里·李和特雷弗·莫赖斯。

Membrane proteins perform a variety of biological functions that are critical to the survival of bacteria, plants, and eukaryotes. Crystal structures of these membrane proteins have shed enormous insights into the functions of transporters, signal transduction receptors, and enzymes. However, crystallization of membrane proteins remains a major bottleneck for structure determination. The use of lipid mesophases, such as lipid cubic phase (LCP), has been highly successful in obtaining high-resolution crystal structures. One of the major factors for successful membrane protein crystallization in LCP is the ability of the protein to diffuse within the lipid bilayer. The diffusion rate of the protein is influenced by protein aggregation, LCP composition, and environment, and can be measured by fluorescence recovery after photobleaching (FRAP). FRAP is able to measure the amount of time required for the fluorescence intensity of a tagged protein to recover within the LCP drop after photobleaching. FRAP allows immediate screening of crystallization conditions, without having to wait weeks to determine whether the crystallization conditions are amenable for crystal growth. The Formulatrix FRAP is a standalone imager capable of high-throughput FRAP to bleach and image a 96-well plate in under 50 minutes. This system will allow rapid screening of LCP lipid components, precipitant composition, ligand and protein modifications. The Formulatrix FRAP system is not available anywhere in Canada and will become an important resource for the entire structural biology community in Toronto. Moreover, the Formulatrix FRAP will expand the current capabilities of an integrated, multi-department UToronto Membrane Protein Crystallization Facility directed by Profs. Jeffrey Lee and Trevor Moraes.