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

• 批准号: 8220753
• 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
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220753
• 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; public health relevance; research study; structural genomics; tool; usability; vapor; wasting

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）：在本项目中，我们建议开发新的仪器来进行脑源性G蛋白偶联受体（GPCR）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测定的高度有序晶体。该仪器（LCPJet）将采用介晶立方相（LCP）蛋白质结晶技术，该技术已被证明是一种非常成功的膜蛋白质结晶方法，通过喷墨点胶提供皮升体积的蛋白质和沉淀剂，为这些现有技术创造了一种新的方法。内消旋LCP创造了一个组织和稳定的环境，其中蛋白质保持其天然构象和活性，从而更好地促进柠檬酸盐的成核和晶体生长以使膜蛋白结晶。与使用纳升体积的其他方法相比，通过LCPJet的皮升液滴的喷墨分配将需要小一个数量级的体积。这将扩展晶体学家有限的GPCR样品的使用，从而能够通过更多数量的结晶实验探索更大的结晶参数空间，同时使用更少的材料。LCPJet将进行前所未有的熔融脂质直接与蛋白质溶液的即时混合。这种能力将消除熔融脂质和蛋白质溶液的机械混合，并将提供使用亚纳升体积的不同脂质和配体添加剂的筛选，从而推进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。这种方法消除了具有单独的液体处理机器人来制定优化屏幕的要求，从而节省了时间并降低了费用。LCPJet将为用户提供灵活性，以执行海绵，bicelle，蒸汽扩散和微批量筛选实验。获得LCPJet技术将为GPCR的晶体发生提供更有效的方法，从而产生更多的高分辨率晶体结构，从而导致开发更有选择性的药物治疗方法，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，其使用高效的介观脂质立方相（LCP）方法对脑源性GPCR进行超低体积结晶筛选。最终，这一工具将加快GPCR的结构测定，从而有益于生物学和医学，并改善患者的心理健康。