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

膜蛋白的片上结晶和原位 X 射线分析

• 批准号: 7794997
• 负责人: Paul J. A. Kenis
• 金额: $ 33.82万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7794997
• 关键词: Address; Agreement; Anemia; Artificial Membranes; Behavior; Biological Process; Cataract; Cell Respiration; Cirrhosis; Copper; Crystallization; Data; Databases; Deposition; Detergents; Diabetes Mellitus; Disease; Drug Delivery Systems; Drug Design; Environment; Enzymes; Epilepsy; Excision; Exhibits; Family member; G-Protein-Coupled Receptors; Genomics; Growth; Heme; Hereditary Disease; Human; Human Genetics; Hypertension; Hypertrophic Cardiomyopathy; In Situ; Integral Membrane Protein; Knowledge; Leigh Disease; Libraries; Light; Link; Lipids; Lung diseases; Mediator of activation protein; Medical; Membrane; Membrane Proteins; Methodology; Methods; Microfluidics; Molecular Conformation; Muscle Rigidity; Nature; Oxidases; Oxygen; Phase; Play; Probability; Process; Production; Proteins; Research Methodology; Resolution; Respiration; Roentgen Rays; Role; Sampling; Screening procedure; Solutions; Structure; System; Temperature; Testing; United States National Institutes of Health; Water; X ray diffraction analysis; X-Ray Diffraction; copper oxidase; cryogenics; deafness; design; fighting; high throughput screening; improved; leukodystrophy; liver cystic fibrosis; member; nanolitre scale; pathogenic bacteria; prevent; protein expression; protein function; protein structure; public health relevance; research study; respiratory; success; three dimensional structure

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.

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 摘要 膜蛋白作为跨细胞和细胞内边界的物质和信息的介质，在许多生物过程中发挥着重要作用。许多疾病都与膜蛋白的功能障碍有关，但只有了解蛋白质的 3D 结构，才能合理设计治疗方法。尽管膜蛋白 (MP) 在许多生物过程以及许多疾病中发挥着重要作用，但膜蛋白 (MP) 的结构特征却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变得复杂。该项目将推进用于 MP 内观结晶的微流体平台。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。该家族的氧还原成员对于细胞呼吸（一种重要的生物功能）至关重要。它们的功能障碍会导致细胞能量产生不足，这与多种人类遗传疾病有关。这种有前途的内消旋方法通过将 MP 维持在由脂质中间相组成的人工膜样环境中，防止 MP 失去其天然构象，晶体可以直接从该环境中生长。我们将开发微流体芯片，实现纳升级内消旋 MP 结晶以及随后对形成的 MP 晶体进行片上原位 X 射线分析。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。与此同时，我们将开发微流控芯片，用于快速筛选脂质/水系统的相行为，以确定它们是否适合内观结晶。用于 MP 内观结晶的大量脂质将提供更宽的参数空间，可以筛选合适的晶体成核和生长条件。两者相关的努力都有望提高膜蛋白结构测定的速度。具体目标1：开发并应用集成微流控芯片，用于血红素铜呼吸氧化酶的内消旋结晶筛选和原位X射线结构测定。所提出的多室结晶平台将能够（i）每次测试使用 <5 nL MP 溶液筛选合适的结晶条件，（ii）通过原位 X 射线分析进行片上晶体质量筛选，以及（iii）片上采集高分辨率数据，用于在低温条件下确定最有前途的晶体的 MP 结构，而无需对通常敏感的 MP 晶体进行片外处理。具体目标 2：开发并应用集成微流控芯片，以高通量测定（通过 X 射线衍射）用于 MP 内观结晶的脂质的相行为。所提出的多室平台将能够配制一系列不同的中间相成分，用于在一定温度范围内进行 X 射线分析。这些芯片还可以快速研究各种污染物（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。公共健康相关性：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了解有可能促进人类相关遗传疾病的医学治疗。