CRYSTALLIZATION OF AN ABC TRANSPORTER

麦芽糖转运蛋白是ABC（ATP结合盒）的成员 超家族，负责ATP偶联的麦芽糖跨E. coli细胞质膜。 超家族的成员可以在 原核以及真核细胞系统。许多这些 转运蛋白已被证明是正常细胞的关键 这些蛋白质的功能和缺陷与许多 囊性纤维化等疾病。 麦芽糖转运蛋白是一个大的 由三种不同多肽组成的多亚基蛋白复合物， 由一个MalG、一个MalF和两个MalK组成。 申请人已经 开发了一种提纯方法， 活性麦芽糖 目前的建议是确定 麦芽糖转运蛋白的结晶条件， 适合于后续晶体结构的晶体 保持战略定力 这将需要（1）蛋白质的表征 在各种洗涤剂中，（2）均质麦芽糖转运蛋白的产生 （3）溶液的研究， 洗涤剂在聚乙二醇和其它溶剂中的相行为 沉淀剂;（4）蛋白质在 所选择的使用结晶基质的洗涤剂通常 与水溶性蛋白质的结晶有关，和（5） 使用添加剂如 两亲物、二价阳离子和其它具有高 临界胶束浓度将对晶体进行评价， 衍射质量和测试与各种冷冻保护剂的低温， 结晶学 目标是获得高质量的晶体， 为后续的高分辨率开发快速冷冻条件 结构测定，以提供分子基础 了解麦芽糖转运蛋白的功能机制。 这 结构应该揭示控制分子的一般原则 ABC超家族成员的机制，并提供深入了解 疾病的结构基础与缺陷， 运输机

Maltose transporter is a member of the ABC (ATP-binding cassette) superfamily, responsible for ATP coupled transport of maltose across E. coli cytoplasmic membranes. Members of the superfamily can be found in prokaryotic as well as eukaryotic cell systems. Many of these transporters have been demonstrated to be critical for normal cell functions, and defects in these proteins have been related to a number of diseases such as cystic fibrosis. Maltose transporter is a large multi-subunit protein complex formed by three different polypeptides, consisting of one MalG, one MalF and two MalK. The applicant has developed a purification method that yields milligram quantities of active maltose. The current proposal is to determine the crystallization conditions of maltose transporter in order to produce crystals that are suitable for subsequent crystallographic structure determination. This will require (1) characterization of the protein in various detergents, (2) production of homogeneous maltose transporter in milligram quantities selected detergents, (3) the study of solution phase behavior of the detergent in polyethylene glycols and other precipitating agents, (4) the crystallization trials of the protein in selected detergents using the crystallization matrice typically associated with the crystallization of water-soluble proteins, and (5) the refinement of crystallization conditions using additives such amphiphiles, divalent cations and other detergents that have a high critical micellar concentration. Crystals will be evaluated for diffraction quality and tested with various cryo-protectants for cryo- crystallography. The goal is to obtain high quality crystals and to develop flash-freezing conditions for subsequent high resolution structure determination in order to provide the molecular basis for understanding the functional mechanism of maltose transporter. This structure should reveal general principles governing the molecular mechanisms of members of the ABC superfamily, and provide insight into the structural basis of diseases associated with defects in the transporters.