On-Chip Crystallization and In Situ X-ray Analysis of Membrane Proteins

描述（由申请人提供）：膜蛋白的片上结晶和原位x射线分析总结膜蛋白在许多生物过程中发挥重要作用，作为物质和信息跨越细胞和细胞内边界的介质。许多疾病都与膜蛋白的功能障碍有关，但只有在蛋白质的三维结构已知的情况下，才能进行合理的医疗设计。尽管膜蛋白（MPs）在许多生物过程中发挥作用，因此在许多疾病中发挥作用，但其结构表征明显落后于可溶性蛋白。MPs的两亲性使x射线质量晶体的生长变得复杂，便于结构分析。该项目将推进微流控平台的中介观结晶MPs。这些平台将用于解析血红素-铜氧化酶超家族成员的结构和功能。这个家族的氧还原成员对细胞呼吸至关重要，这是一种基本的生物功能。它们的功能失调会导致细胞能量产生不足，这与几种人类遗传疾病有关。有希望的中介观方法通过将MPs维持在由脂质中间相组成的人工膜样环境中来防止它们失去其天然构象，从中可以直接生长晶体。我们将开发微流控芯片，使纳米级中观MP结晶以及随后的片上，原位x射线分析形成的MP晶体。除了将每次测试所需的MP样品量减少到<5 nL外，这种微流体方法还将消除在结晶和x射线分析之间通常高度敏感的MP晶体的直接处理。同时，我们将开发微流控芯片，用于快速筛选脂质/水体系的相行为，以确定它们是否适合中介晶化。大量的脂质用于MPs的介观结晶将提供更广泛的参数空间，可以筛选合适的晶体成核和生长条件。这两方面的工作都有望提高膜蛋白结构的测定率。具体目标1：开发和应用集成微流控芯片用于血红素-铜呼吸氧化酶的介观结晶筛选和原位x射线结构测定。提议的多室结晶平台将实现(i)筛选合适的结晶条件，每次测试使用<5 nL的MP溶液，（ii）通过原位x射线分析进行片上晶体质量筛选，以及（iii）片上获取高分辨率数据，用于在低温条件下确定最有希望的晶体的MP结构，所有这些都不需要对通常敏感的MP晶体进行片外处理。具体目标2：开发和应用集成微流控芯片，用于高通量测定（通过x射线衍射）用于MPs中介观结晶的脂质相行为。提议的多室平台将能够在一定温度范围内制定一系列不同的中间相组成进行x射线分析。这些芯片还将允许快速研究各种污染物的影响，例如通常用于膜蛋白分离的少量洗涤剂，对脂/水相行为的影响。公共卫生相关性：本项目有助于研究膜蛋白结构，并间接阐明其功能。血红素-铜氧化酶与细胞呼吸系统疾病有关，是常见的药物靶点。提高我们对这些蛋白质的理解有可能推进人类相关基因决定疾病的医学治疗。

DESCRIPTION (provided by applicant): On-chip crystallization and in situ X-ray analysis of membrane proteins Summary Membrane Proteins play an important role in many biological processes as mediators of material and information across cellular and intracellular boundaries. Many diseases have been connected to the malfunction of membrane proteins but rational design of medical treatments can only occur once the 3D structure of a protein is known. Despite their role in many biological processes, and thus many diseases, the structural characterization of membrane proteins (MPs) has lagged significantly behind those of soluble proteins. The amphiphilic nature of MPs complicates growth of X-ray quality crystals for structural analysis. This project will advance microfluidic platforms for the in-meso crystallization of MPs. These platforms will be applied to resolve the structure and function of members of the heme-copper oxidase superfamily. The oxygen reducing members of this family are critical in cellular respiration, an essential biological function. Their malfunction can result in insufficient cellular energy production, which has been linked to several human genetic diseases. The promising in-meso approach prevents MPs from loosing their native conformation by maintaining them in an artificial membrane-like environment comprised of lipidic mesophases, from which crystals can be grown directly. We will develop microfluidic chips enabling nanoliter scale in-meso MP crystallization as well as subsequent on-chip, in situ X-ray analysis of MP crystals formed. In addition to reducing the amount of MP sample needed to <5 nL per test, this microfluidic approach will also eliminate direct handling of the often highly sensitive MP crystals between crystallization and X-ray analysis. In parallel, we will develop microfluidic chips for rapid screening of the phase behavior of lipid/water systems for their suitability for in-meso crystallization. A larger number of lipids for in-meso crystallization of MPs will provide a wider parameter space that can be screened for suitable crystal nucleation and growth conditions. Both, related efforts are expected to enhance the rate of membrane protein structure determination. Specific Aim 1: Develop and apply integrated microfluidic chips for in-meso crystallization screening and in situ X-ray structure determination of heme-copper respiratory oxidases. The proposed multi- compartment crystallization platforms will enable (i) screening for suitable crystallization conditions using <5 nL of MP solution per test, (ii) on-chip crystal quality screening via in situ X-ray analysis, as well as (iii) on-chip acquisition of high resolution data for MP structure determination of the most promising crystals under cryogenic conditions, all without off-chip handling of the often sensitive MP crystals. Specific Aim 2: Develop and apply integrated microfluidic chips for high throughput determination (via X-ray diffraction) of the phase behavior of lipids intended for the in-meso crystallization of MPs. The proposed multi-compartment platforms will be capable of formulating a range of different mesophase compositions for X-ray analysis over a range of temperatures. These chips will also allow for rapid study of the effects of various contaminants, such as small amounts of the detergents typically used in membrane protein isolation, on lipid/water phase behavior. PUBLIC HEALTH RELEVANCE: This project contributes to the study of membrane protein structure and indirectly to the elucidation of their function. Heme-copper oxidases have been linked to cellular respiratory diseases and are common drug targets. Improving our understanding of these proteins has the potential to advance medical treatment of the related genetically determined diseases in humans.