Novel capture reagents for membrane protein structure determination

用于膜蛋白结构测定的新型捕获试剂

• 批准号: 8150337
• 负责人: Lan Guan
• 金额: $ 29.7万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8150337
• 关键词: 3-Dimensional; Address; Affinity; Ankyrin Repeat; Binding; Binding Proteins; Biochemical; Carrier Proteins; Cell physiology; Complex; Crystallization; Cytochromes; Data; Disease; Enzymes; Erythrocytes; Escherichia coli; Funding; Glucose; Goals; Health; Human; In Situ; In Vitro; Interdisciplinary Study; Knowledge; Libraries; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Methods; Micrococcal Nuclease; Molecular; Molecular Chaperones; Monoclonal Antibodies; Play; Principal Investigator; Probability; Process; Production; Proteins; Reagent; Relative (related person); Reporting; Research; Resolution; Ribosomes; Roentgen Rays; Role; SLC2A1 gene; Sampling; Scaffolding Protein; Screening procedure; Shapes; Specificity; Structure; Testing; Thioredoxin; United States National Institutes of Health; X-Ray Crystallography; base; combinatorial; conformer; experience; glucose transport; improved; in vitro testing; in vivo; insight; lactose permease; melibiose permease; novel; novel strategies; permease; programs; protein complex; protein structure; public health relevance; success; sugar; tool

DESCRIPTION (provided by applicant): There is an urgent need to develop new tools to facilitate crystallization of membrane proteins. The long-term goal of this application is to understand the molecular mechanisms of physiologically important and disease-related membrane proteins by X-ray crystallographic determination. The overall objective, which is an important step towards attainment of our long-term goal, is to test a novel approach -- the production and use of membrane protein capture reagents to facilitate crystallization and structure determination of membrane transporters. Although successful application of capture reagents as crystallization chaperones for structure determination of membrane proteins has been reported, the approach has not been widely utilized. We also suggest that novel molecules capable of trapping weakly bound but physiologically important membrane protein complexes in situ will be discovered. In this proposal, scaffold proteins-based combinatorial libraries will be constructed and tested by in vitro ribosome displays and in vivo functional screening to discover specific capture reagents that bind to the bacterial melibiose permease (MelB) or to the human red blood cell glucose transport (GLUT1). A proof-of-principle study on a capture reagent for trapping weakly interacting protein-membrane protein complexes will be carried out using well-characterized lactose permease (LacY) or MelB bound to their regulatory enzyme IIAglc. Specificity and affinity of selected capture reagents will be evaluated by biochemical and biophysical methods. Furthermore, selected capture reagents will also be tested for co-crystallization with their targeted membrane permeases. This will expedite the crystal structure determination of MelB. The impact of this proposal is that the methodology established will serve as a general tool for membrane protein structure determination, as well as functional studies, and the novel high-resolution structure of MelB will advance our knowledge of function at the atomic level. The proposed research is significant because the approach proposed addresses a critical barrier to progress in the field of membrane protein structure determination. PUBLIC HEALTH RELEVANCE: Membrane proteins play crucial roles in many aspects of cell function and are the main targets of pharmacologically and toxicologically active substances. In comparison to soluble proteins, the number of available membrane protein structures is very limited, and this lack of high-resolution structures precludes deeper insights into essential aspects of human health and disease. The approach proposed here can serve as a general tool to enhance the probability of crystallization of membrane proteins and thereby facilitate their structure determination.

描述（由申请人提供）：迫切需要开发新的工具来促进膜蛋白的结晶。该应用的长期目标是通过X射线晶体学测定来了解生理重要和与疾病相关的膜蛋白的分子机制。总体目标是实现我们长期目标的重要一步，是测试一种新方法 - 膜蛋白捕获试剂的生产和使用，以促进膜转运蛋白的结晶和结构测定。尽管已经报告了捕获试剂作为结晶伴侣在膜蛋白结构测定中的成功应用，但尚未广泛使用该方法。我们还建议将发现能够捕获弱结合但生理上重要的膜蛋白复合物的新分子。在此提案中，将通过体外核糖体显示和体内功能筛选来构建和测试基于支架蛋白的组合文库，以发现与细菌蜜饯渗透酶（MERB）或人类红色血细胞葡萄糖转运（Glut1）结合的特定捕获试剂（MELB）。针对捕获试剂的原则证明研究将使用特征良好的乳糖粘酶（蕾丝）或与其调节酶IIAGLC结合的梅尔布进行捕获弱相互作用的蛋白质蛋白蛋白复合物。选定捕获试剂的特异性和亲和力将通过生化和生物物理方法评估。此外，选定的捕获试剂还将通过其靶向膜磁盘进行共结晶测试。这将加快MELB的晶体结构测定。该建议的影响是，建立的方法将作为膜蛋白结构确定以及功能研究的一般工具，而新型MELB的高分辨率结构将提高我们在原子水平上的功能知识。拟议的研究很重要，因为该方法解决了膜蛋白结构测定领域进展的关键障碍。 公共卫生相关性：膜蛋白在细胞功能的许多方面起着至关重要的作用，是药理和毒理学活性物质的主要靶标。与可溶性蛋白相比，可用的膜蛋白结构的数量非常有限，这种缺乏高分辨率结构排除了对人类健康和疾病基本方面的更深入的见解。此处提出的方法可以用作一种一般工具，以增强膜蛋白结晶的可能性，从而促进其结构的确定。