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。该应用程序的长期目标是通过x射线晶体学测定了解生理上重要的和与疾病相关的膜蛋白的分子机制。总体目标，这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确定。虽然已有报道成功地将捕获试剂作为结晶伴侣用于膜蛋白结构测定，但该方法尚未得到广泛应用。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。在本研究中，我们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，以发现与细菌美利二糖渗透酶（MelB）或人类红细胞葡萄糖转运（GLUT1）结合的特异性捕获试剂。对捕获弱相互作用蛋白-膜蛋白复合物的捕获试剂的原理验证研究将使用特性良好的乳糖渗透酶（LacY）或MelB结合其调节酶IIAglc进行。所选捕获试剂的特异性和亲和力将通过生化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物的共结晶。这将加快MelB晶体结构的测定。这一建议的影响是，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推进我们在原子水平上的功能知识。提出的研究是重要的，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。

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.