Novel capture reagents for membrane protein structure determination

描述(申请人提供)：迫切需要开发新的工具来促进膜蛋白的结晶。这一应用的长期目标是通过X射线结晶学测定来了解生理上重要的和与疾病相关的膜蛋白的分子机制。总体目标是测试一种新的方法--生产和使用膜蛋白捕获剂，以促进膜转运体的结晶和结构确定，这是实现我们长期目标的重要一步。虽然已有报道成功地将捕捉剂作为结晶伴侣应用于膜蛋白的结构测定，但这种方法还没有得到广泛的应用。我们还认为，能够原位捕获弱结合但具有重要生理意义的膜蛋白复合体的新分子将被发现。在这项计划中，基于支架蛋白的组合文库将通过体外核糖体展示和体内功能筛选来构建和测试，以发现与细菌蜜二糖渗透酶(Melb)或与人红细胞葡萄糖运输(GLUT1)结合的特异性捕获剂。利用具有良好特性的乳糖渗透酶(Lacy)或与其调节酶IIAglc结合的Melb，将对捕获弱相互作用的蛋白质-膜蛋白质复合体的捕捉剂进行原理证明研究。所选捕获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，选定的捕集试剂还将与其目标膜渗透系数进行共结晶测试。这将加快MALB的晶体结构测定。这一提议的影响是，所建立的方法学将作为膜蛋白质结构测定以及功能研究的通用工具，而Melb的新的高分辨率结构将促进我们在原子水平上对功能的了解。这项研究具有重要意义，因为所提出的方法解决了膜蛋白质结构测定领域取得进展的一个关键障碍。 与公共健康相关：膜蛋白在细胞功能的许多方面起着关键作用，是药理和毒理活性物质的主要靶标。与可溶性蛋白质相比，可用的膜蛋白结构的数量非常有限，这种高分辨率结构的缺乏阻碍了对人类健康和疾病的基本方面的更深入的了解。本文提出的方法可以作为一种通用工具来提高膜蛋白的结晶几率，从而促进其结构的确定。

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.