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

用于 GPCR 脂质立方相晶体筛选的喷墨系统

基本信息

批准号：
8056712
负责人：
Patrick W Cooley
金额：
$ 35万
依托单位：
MICROFAB TECHNOLOGIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8056712
关键词：
Address Adenosine A2A Receptor Adrenergic Receptor Advanced Development Aspirate substance Biological Assay Biology Bolus Infusion Brain Complex Crystallization Crystallography Custom Detergents Development Diffusion Drops Drug Formulations Environment G-Protein-Coupled Receptors Generations Glass Goals Government Growth Health Human Humidity Joints Lead Ligands Lipids Liquid substance Maintenance Marketing Mechanics Medical Medicine Membrane Proteins Mental Health Methodology Methods Micelles Modeling Molecular Conformation Molecular Structure Monoglycerides Patients Phase Porifera Process Proteins Research Institute Resolution Robot Sampling Screening procedure Site Societies Solutions Source Structure System Techniques Technology Temperature Testing Time Validation X ray diffraction analysis X-Ray Diffraction base combinatorial cost design design and construction drug discovery flexibility fly improved innovation instrument instrumentation nanolitre novel operation protein aggregation prototype research study structural genomics tool usability vapor wasting

项目摘要

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.

描述（由申请人提供）：在此项目中，我们建议开发新的仪器，以对大脑衍生的G蛋白偶联受体（GPCR）和其他膜蛋白进行结晶筛选，以获得高度有序的晶体，以进行X射线衍射和分子结构的测定。该仪器（LCPJET）将采用脂质立方相（LCP）蛋白质结晶技术，证明是一种非常成功的膜蛋白结晶方法，并通过Inkjet分配蛋白质和沉淀剂的PICOLITER体积递送，从而为这些现有技术创造了一种新的方法论。在中液中，LCP创建了一个组织和稳定的环境，在该环境中，蛋白质保留其天然构象和活性，从而更好地促进了顽固的成核和晶体生长，以结晶膜蛋白质。与使用纳米尺量的其他方法相比，LCPJET对PICOLITER滴剂的喷墨分配量的数量级要少。这将扩展晶体学家有限的GPCR样品的使用，从而通过更多的结晶实验来探索较大的结晶参数空间，同时使用较少的材料。 LCPJET将直接与蛋白质溶液直接执行熔融脂质的前所未有的混合。该能力将消除熔融脂质和蛋白质溶液的机械混合，并将使用亚纳维剂量筛选不同的脂质和配体添加剂，从而提高LCP结晶径的可行性。该系统还将能够使用喷墨分配来实现自定义优化 /网格屏幕结晶试验。这种方法消除了拥有单独的液体处理机器人以制定优化屏幕的要求，从而节省了时间并减少了费用。 LCPJET将提供用户的灵活性，以执行海绵，bicelle，蒸气扩散和微匹配筛选实验。进入LCPJET技术将为GPCR的结晶提供一种更有效的方法，从而导致更多的高分辨率晶体结构，从而导致发展更具选择性的药理治疗方法，以改善心理健康。公共卫生相关性：LCPJET是一种基于喷墨的仪器，使用在中脂立方体（LCP）方法中使用高效的大脑衍生GPCR进行超低体积结晶筛选。最终，该工具将加快GPCR的结构确定，从而使生物学和医学受益，并改善患者的心理健康。