Microfluidic Membrane Protein Crystallization for HIgh Resolution Proteomics

描述（由申请人提供）：膜蛋白负责生物学中的关键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障碍有关，但只有在知道蛋白质的3D结构后，才能合理设计医疗方法。然而，膜蛋白的两亲性使高质量晶体的生长复杂化，用于通过X射线衍射进行结构分析。此外，其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。在蛋白质数据库中存放的> 47，000个结构中，不到300个是膜蛋白，并且其中只有一小部分是人类膜蛋白，尽管基因组估计约30%的蛋白质预计是整合的膜蛋白。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，这种差异代表了开发结晶新方法的关键需求领域。我建议阐明的结构和运作机制的某些成员的一类血红素铜氧化酶膜蛋白。该蛋白质家族的氧还原成员是细胞呼吸过程中的关键酶。许多人类遗传性疾病与氧化酶功能障碍有关，呼吸氧化酶有可能成为致病菌的药物靶点。微量结晶方法将能够确定合适的结晶条件，同时使用微量的蛋白质（nL至pL而不是uL规模）并将蛋白质保持在膜样环境中。具体目标1。完成我们的微流控结晶平台的设计、制造和验证，该平台集成了流体计量、混合和沉淀剂添加，通过内孔方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存在于膜状的非晶中间相中，提高了获得高质量晶体的机会。具体目标2。在使用SA 1的微流控平台鉴定介观结晶条件后，阐明血红素-铜呼吸氧化酶膜蛋白超家族的各种细菌成员的结构和生物学机制。更具体地说，该方法将用于（a）改进仅可获得低分辨率结构信息的蛋白质/突变体的结构信息，和（B）获得其结构先前未被解析的新蛋白质构建体或突变体的结构。将进行结构-功能研究，以扩展其功能背后的分子机制的生物学知识。公共卫生相关性这项研究与公共卫生的相关性在于阐明与细胞能量产生相关的各种酶发挥作用的机制，从而促进和加快对与呼吸氧化酶膜蛋白相关的疾病的了解，并指导各种药物的开发治疗。

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.