Enabling Nanoliter Membrane Protein Crystallization(RMI)

描述（由申请人提供）：据估计，超过一半的药物靶点是膜蛋白。需要对膜蛋白结构的详细了解（使用膜蛋白晶体的x射线衍射确定），以了解由这些蛋白质驱动的过程，并合理设计可以作为许多疾病药物治疗的新分子。用于结晶的膜蛋白溶液供应有限，因此结晶试验必须经济小型化，以提高结晶成功率，同时降低每种蛋白质结构的成本。该提案描述了一个多学科的研究项目，旨在开发和验证微流体技术，使膜蛋白能够快速和经济地结晶，在纳米升尺度上进行，直接测试晶体的衍射质量。该计划的具体目标是：(1)开发和验证基本的基于塞子的微流控技术，以便对膜蛋白结晶所需的各种试剂进行准确和轻松的流体操作；(2)使用基本的微流控技术开发基于预加载墨盒的膜蛋白结晶方法；(3)通过以下方式扩大该技术的影响：a)为有前途的新结晶技术开发预加载墨盒，b)确定传统和基于墨盒的技术之间的关系，c)通过完善原位衍射来避免处理晶体时潜在的损坏，d)通过我们的合作者（Scripps和Argonne国家实验室）将该技术传播到膜蛋白晶体界。这些工具将是简单和廉价的设置和操作，并提供给小型实验室。它们还将与自动化和机器人兼容。这项技术将对膜蛋白结晶领域产生重大影响，使个体研究人员和大型研究中心能够快速经济地确定新的膜蛋白靶点的x射线晶体结构。

DESCRIPTION (provided by applicant): It is estimated that over half of drug targets are membrane proteins. Detailed knowledge of membrane protein structure (determined using X-ray diffraction of membrane protein crystals) is needed to understand the processes that are driven by these proteins, and to rationally design new molecules that can serve as drug treatments for many diseases. The supply of membrane protein solution for crystallization is limited, and so crystallization trials must be economically miniaturized in order to increase the success rate of crystallization while reducing the cost of each protein structure. This proposal describes a multi-disciplinary research program that aims to develop and validate microfluidic technology enabling rapid and economical crystallization of membrane proteins, performed on nanoliter scale with direct testing of diffraction quality of crystals. The specific aims of the program are to: (1) develop and to validate basic plug-based microfluidic technology for accurate and easy fluid manipulation of the various reagents required for membrane protein crystallization, (2) to use the basic microfluidic technology to develop a preloaded cartridge-based approach to crystallization of membrane proteins, and (3) to broaden the impact of the technology developed by: a) developing preloaded cartridges for the promising new crystallization techniques, b) determining the relationship between traditional- and cartridge-based techniques, c) avoiding the potential for damage in handling of crystals by perfecting in-situ diffraction, and d) disseminating the technology to the membrane protein crystal community though our collaborators (Scripps and Argonne National Labs). These tools will be simple and inexpensive to set up and operate, and available to small laboratories. They will also be compatible with automation and robotics. This technology will have a significant impact on the field of membrane protein crystallization, enabling both individual investigators and large centers to determine X-ray crystal structures of new membrane protein targets rapidly and economically.