Inkjet Based System for Lipid Cubic Phase Crystal Screening of GPCR's

描述(申请人提供)：在这个项目中，我们建议开发新的仪器来进行脑源性G蛋白偶联受体(GPCRs)和其他膜蛋白的结晶筛选，以获得用于X射线衍射和分子结构确定的高度有序的晶体。这台仪器(LCPJet)将采用脂质立方相(LCP)蛋白质结晶技术，这是一种非常成功的膜蛋白质结晶方法，通过喷墨分配实现蛋白质和沉淀剂的皮升体积输送，为这些现有技术创造了一种新的方法。In Meso LCP创造了一个组织和稳定的环境，使蛋白质保持其天然的构象和活性，从而更好地促进顽固性蛋白质的成核和晶体生长，使膜蛋白结晶。与使用纳升体积的其他方法相比，LCPJet喷墨分配皮升液滴所需的体积要小一个数量级。这将扩大结晶学家有限的GPCR样品的使用范围，从而能够通过更多的结晶实验来探索更大的结晶参数空间，同时使用更少的材料。LCPJet将史无前例地将熔融的脂肪直接与蛋白质溶液混合。这种能力将消除熔融的脂类和蛋白质溶液的机械混合，并将提供使用亚纳升体积筛选不同的脂类和配体添加剂，从而提高LCP结晶试验的可行性。该系统还将能够使用喷墨点胶即时生成定制优化/网格屏幕结晶试验。这种方法省去了单独的液体处理机器人来制定优化筛选的要求，从而节省了时间和成本。LCPJet将为用户提供执行海绵、双孔、蒸汽扩散和微批筛选试验的灵活性。获得LCPJet技术将提供一种更有效的方法来研究GPCRs的晶体形成，从而产生更多的高分辨率晶体结构，从而开发出更多用于改善心理健康的选择性药物治疗。 与公众健康相关：LCPJet是一种基于喷墨技术的仪器，使用高效的中脂立方相(LCP)方法对脑源性GPCRs进行超低容量结晶筛查。最终，这一工具将加快GPCR的结构确定，从而有利于生物学和医学，并改善患者的心理健康。

DESCRIPTION (provided by applicant): In this project, we propose to develop new instrumentation to perform crystallization screening of brain-derived G-protein coupled receptors (GPCRs) and other membrane proteins to obtain highly ordered crystals for X-ray diffraction and molecular structure determination. This instrument (LCPJet) will employ lipidic cubic phase (LCP) protein crystallization technology, demonstrated to be a highly successful membrane protein crystallization approach, with picoliter volume delivery of protein and precipitant via inkjet dispensing, creating a novel methodology for these existing technologies. The in meso LCP creates an organizing and stabilizing environment in which the protein retains its native conformation and activity, thus better facilitating nucleation and crystal growth of recalcitrant to crystallize membrane proteins. The inkjet dispensing of picoliter drops by LCPJet will require an order of magnitude less volume compared to other approaches using nanoliter volumes. This will extend the use of a crystallographer's limited GPCR sample, thus enabling the exploration of larger crystallization parameter space through a greater number of crystallization experiments, while using less material. LCPJet will perform unprecedented on-the-fly mixing of molten lipid directly with the protein solution. This capability will eliminate the mechanical mixing of molten lipid and protein solution and will provide for the screening of different lipids and ligand additives using sub-nanoliter volumes, thus advancing the feasibility of LCP crystallization trails. The system will also be capable of on-the-fly generation of custom optimization / grid screen crystallization trials using inkjet dispensing. This approach eliminates the requirement of having a separate liquid handling robot to formulate the optimization screens, thus saving time and reducing expense. LCPJet will provide the user flexibility to perform sponge, bicelle, vapor diffusion and microbatch screening experiments. Access to the LCPJet technology will offer a more effective approach to the crystallogenesis of GPCR's resulting in a greater number of high-resolution crystal structures, thus leading to development of more selective pharmacologic treatments for improving mental health. PUBLIC HEALTH RELEVANCE: LCPJet is an inkjet-based instrument that performs ultra-low volume crystallization screening of brain-derived GPCRs using the highly effective in meso lipid cubic phase (LCP) approach. Ultimately, this tool will expedite the structural determination of GPCR's, thus benefiting biology and medicine, and improving patient mental health.