Structural Characterization of Endophilin-2

描述(由申请人提供)： 这项研究的长期目标是确定不同结构的内嗜素-A2的晶体结构，这将加强现有的体内和分子生物学研究，涉及复杂和高度调控的细胞内吞作用的机制。亲内蛋白是一个含有SH3结构域的蛋白质家族，在细胞骨架蛋白介导的内吞作用、蛋白质转运、细胞结构维持、细胞增殖和分化以及免疫反应等过程中发挥作用。目前还没有这些蛋白质的结构数据。在这个方案中，我们将从大肠杆菌中纯化重组大鼠亲内毒素-A2，并启动结晶筛选。为了促进蛋白质结晶，我们将通过有限的蛋白质分解来确定稳定的结构域。最后，晶体生长将被优化，以产生衍射良好的晶体，以便进行结构确定研究。结构信息将导致深入了解内亲和素-A2是如何发挥作用的，无论是单独还是与其各种蛋白质结合伙伴。这将增加我们对基本内吞过程的理解，以及这些过程中的变化如何导致疾病状态。

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the crystal structures of various constructs of endophilin-A2, which will augment existing in vivo and molecular biology studies of the mechanisms involved in the complex and highly regulated cellular process of endocytosis. Endophilins are an SH3 domain containing family of proteins that function in processes such as clathrin-mediated endocytosis, protein trafficking, cell architecture maintenance, cell proliferation and differentiation, and in the immune response. There are no available structural data for these proteins. In this proposal we will purify recombinant rat endophilin-A2 from E. coli and initiate crystallization screens. To facilitate protein crystallization we will identify stable domains by limited proteolysis. Finally, crystal growth will be optimized to generate welldiffracting crystals to allow for structural determination studies. Structural information will lead to insights into how endophilin-A2 functions, both alone and with its various protein binding partners. This will increase our understanding of basic endocytic processes, and how alterations in these processes can result in disease states.