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: 8311706
• 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/8311706
• 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 射线晶体学测定了解生理上重要的和疾病相关的膜蛋白的分子机制。总体目标是测试一种新方法，即生产和使用膜蛋白捕获试剂以促进膜转运蛋白的结晶和结构测定，这是实现我们长期目标的重要一步。尽管已有报道成功应用捕获试剂作为结晶伴侣来确定膜蛋白的结构，但该方法尚未得到广泛应用。我们还建议将发现能够原位捕获弱结合但生理上重要的膜蛋白复合物的新分子。在该提案中，将构建基于支架蛋白的组合文库，并通过体外核糖体展示和体内功能筛选进行测试，以发现与细菌蜜二糖通透酶（MelB）或人红细胞葡萄糖转运（GLUT1）结合的特异性捕获试剂。将使用已充分表征的乳糖通透酶 (LacY) 或与其调节酶 IIAglc 结合的 MelB，对用于捕获弱相互作用的蛋白质-膜蛋白复合物的捕获试剂进行原理验证研究。所选捕获试剂的特异性和亲和力将通过生物化学和生物物理方法进行评估。此外，还将测试选定的捕获试剂与其目标膜渗透酶的共结晶。这将加快 MelB 晶体结构的测定。该提案的影响在于，所建立的方法将作为膜蛋白结构测定和功能研究的通用工具，而 MelB 的新型高分辨率结构将增进我们对原子水平功能的了解。这项研究意义重大，因为所提出的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发挥着至关重要的作用，并且是药理学和毒理学活性物质的主要目标。与可溶性蛋白质相比，可用的膜蛋白结构的数量非常有限，并且缺乏高分辨率结构阻碍了对人类健康和疾病的基本方面的更深入的了解。这里提出的方法可以作为增强膜蛋白结晶概率的通用工具，从而促进其结构测定。