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

描述（由申请人提供）：膜蛋白的芯片上结晶和原位X射线分析概述膜蛋白在许多生物过程中作为跨越细胞和细胞内边界的物质和信息的介体发挥重要作用。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。尽管它们在许多生物过程中起作用，因此在许多疾病中，膜蛋白（MP）的结构表征明显落后于可溶性蛋白。MP的两亲性质使用于结构分析的X射线质量晶体的生长复杂化。该项目将推进微流体平台，用于MP的介观结晶。这些平台将被应用于解决血红素铜氧化酶超家族成员的结构和功能。该家族的氧还原成员在细胞呼吸中至关重要，这是一种基本的生物功能。它们的功能障碍可能导致细胞能量产生不足，这与几种人类遗传疾病有关。有前途的中介方法通过将MP保持在由介晶中间相组成的人工膜状环境中来防止MP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，使纳升规模的介观MP结晶，以及随后的芯片上，在原位X射线分析MP晶体形成。除了将每次测试所需的MP样品量减少到<5 nL之外，这种微流体方法还将消除在结晶和X射线分析之间对通常高度敏感的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，以确定其是否适合于介观结晶。用于MP的介观结晶的更大数量的脂质将提供更宽的参数空间，其可以针对合适的晶体成核和生长条件进行筛选。这两方面的努力都有望提高膜蛋白结构测定的速度。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。所提出的多隔室结晶平台将使得能够（i）使用每次测试<5 nL的MP溶液筛选合适的结晶条件，（ii）通过原位X射线分析进行芯片上晶体质量筛选，以及（iii）在芯片上获取高分辨率数据，用于在低温条件下确定最有前途的晶体的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。具体目标二：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）的相行为的脂质打算在介观结晶的MP。所提出的多隔室平台将能够在一定温度范围内配制用于X射线分析的一系列不同中间相组合物。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去污剂对脂质/水相行为的影响。公共卫生相关性：该项目有助于膜蛋白结构的研究，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促进人类相关遗传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.