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各种结构的endophilin-A2的晶体结构，这将增加现有的体内和分子生物学研究的机制，涉及复杂的和高度调节的细胞内吞过程。内亲蛋白是一种含有SH 3结构域的蛋白质家族，其在诸如网格蛋白介导的内吞作用、蛋白质运输、细胞结构维持、细胞增殖和分化以及免疫应答等过程中起作用。这些蛋白质没有可用的结构数据。本研究拟从大肠杆菌中纯化重组大鼠endophilin-A2。大肠杆菌，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水解来鉴定稳定结构域。最后，晶体生长将被优化，以产生良好的衍射晶体，以允许结构测定研究。结构信息将导致深入了解endophilin-A2的功能，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，以及这些过程的改变如何导致疾病状态。

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.