Novel capture reagents for membrane protein 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。这项应用的长期目标是通过X射线晶体学测定来了解生理学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的方法-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。虽然已经报道了捕获试剂作为膜蛋白结构测定的结晶分子伴侣的成功应用，但是该方法尚未被广泛使用。我们还建议，新的分子能够捕获弱结合，但生理上重要的膜蛋白复合物原位将被发现。在这个提议中，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蛋白（GLUT 1）结合的特异性捕获试剂。将使用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蛋白-膜蛋白复合物的捕获试剂进行原理验证研究。将通过生物化学和生物物理方法评价所选捕获试剂的特异性和亲和力。此外，还将测试所选捕获试剂与其靶向膜渗透酶的共结晶。这将加快MelB的晶体结构测定。这个建议的影响是，所建立的方法将作为膜蛋白结构测定以及功能研究的通用工具，并且MelB的新的高分辨率结构将在原子水平上推进我们对功能的认识。拟议的研究是重要的，因为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起着至关重要的作用，并且是毒性和毒理学活性物质的主要靶点。与可溶性蛋白相比，可用的膜蛋白结构的数量非常有限，并且这种高分辨率结构的缺乏阻碍了对人类健康和疾病的重要方面的更深入了解。这里提出的方法可以作为一个通用的工具，以提高膜蛋白的结晶的概率，从而促进其结构的测定。

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.