Microfluidic Membrane Protein Crystallization for HIgh Resolution Proteomics

描述（由申请人提供）：膜蛋白在生物学中负责关键的细胞信号传导和物质/能量转导过程。许多疾病都与膜蛋白的功能失调有关，但只有在了解蛋白质的三维结构后，才能进行合理的医疗设计。然而，膜蛋白的两亲性使高质量晶体的生长变得复杂，难以通过x射线衍射进行结构分析。此外，它们有限的可用性阻碍了高通量筛选工作以确定合适的结晶条件。在蛋白质数据库中储存的47,000个结构中，只有不到300个是膜蛋白，而其中只有一小部分是人类膜蛋白，尽管基因组估计-30%的蛋白质预计是完整的膜蛋白。考虑到膜蛋白在与许多疾病相关的途径中发挥的关键作用，这种差异代表了迫切需要开发新的结晶方法的领域。我建议阐明一类血红素-铜氧化酶膜蛋白的某些成员的结构和运作机制。该蛋白家族的氧还原成员是细胞呼吸过程中的关键酶。许多人类遗传疾病与氧化酶功能障碍有关，呼吸道氧化酶有可能成为致病菌的药物靶点。微尺度结晶方法可以确定合适的结晶条件，同时使用极少量的蛋白质（nL到pL而不是uL尺度）并将蛋白质保持在膜状环境中。具体目标完成我们的微流体结晶平台的设计、制造和验证，该平台集成了流体计量、混合和沉淀剂添加，通过介观法筛选潜在的结晶条件。在这种方法中，蛋白质在结晶过程中驻留在膜状的脂质中间期，从而增加了获得高质量晶体的机会。具体目标2。利用SA1微流控平台鉴定中介观结晶条件，阐明血红素-铜呼吸氧化酶膜蛋白超家族各细菌成员的结构和生物学机制。更具体地说，该方法将应用于(a)改善只有低分辨率结构信息可用的蛋白质/突变体的结构信息，以及(b)获得以前未解析结构的新蛋白质构建物或突变体的结构。结构功能研究将进行，以扩大其功能背后的分子机制的生物学知识。本研究对公共卫生的意义在于阐明与细胞能量产生功能相关的各种酶的作用机制，从而促进和加快对呼吸氧化酶膜蛋白相关疾病的认识，指导各种药物治疗的发展。

DESCRIPTION (provided by applicant): Membrane proteins are responsible for key cell signaling and material/energy transduction processes in biology. Many diseases have been connected to the malfunction of membrane proteins but the rational design of medical treatments can only occur once the 3D structure of a protein is known. However, the amphiphilic nature of membrane proteins complicates the growth of high quality crystals for structural analysis by X-ray diffraction. Moreover, their limited availability hampers high-throughput screening efforts to determine suitable crystallization conditions. Of the >47,000 structures deposited in the Protein Databank less than 300 are for membrane proteins, and of these only a tiny fraction are human membrane proteins, despite genomic estimates that -30% of proteins are expected to be integral membrane proteins. This disparity represents an area of critical need for the development of new methods for crystallization given the key role that membrane proteins play in pathways related to many diseases. I propose the elucidation of the structure and operational mechanism of certain members of a class of heme-copper oxidase membrane proteins. The oxygen reducing members of this family of proteins are critical enzymes in the respiration process of the cell. A number of human genetic diseases have been tied to oxidase malfunction, and respiratory oxidases have potential as drug targets for pathogenic bacteria. A microscale crystallization method will enable determination of suitable crystallization conditions while using miniscule amounts of protein (nL to pL instead of uL scale) and maintaining the protein in a membrane-like environment. Specific Aim 1. Finalize the design, fabrication, and validation of our microfluidic crystallization platforms that integrate fluid metering, mixing, and precipitant addition to screen for potential crystallization conditions by the in-meso method. In this method, the protein resides in a membrane-like, lipidic mesophase during crystallization, enhancing the chance of obtaining a high quality crystal. Specific Aim 2. Elucidate the structure and biological mechanisms of various bacterial members of the heme-copper respiratory oxidase superfamily of membrane proteins after using the microfluidic platforms of SA1 to identify in-meso crystallization conditions. More specifically, this method will be applied to (a) improve the structural information on proteins/mutants of which only low resolution structural information is available and (b) obtain the structure of novel protein constructs or mutants of which the structure has not previously been resolved. Structure-function studies will be performed in order to extend biological knowledge of the molecular mechanism behind their function. PUBLIC HEALTH RELEVANCE The relevance of this research to public health lies in the elucidation of the mechanism whereby various enzymes associated with cellular energy production function, thus facilitating and expediting the understanding of diseases associated with respiratory oxidase membrane proteins, and guiding the development of various pharmaceutical treatments.