CRYSTALLIZATION OF AN ABC TRANSPORTER

麦芽糖转运体是ABC(三磷酸腺苷结合盒)的成员 负责麦芽糖的ATP偶联转运。 Coli细胞质膜。超级大家庭的成员可以在 原核生物和真核细胞系统。其中许多 转运蛋白已被证明对正常细胞至关重要 这些蛋白质的功能和缺陷与许多 囊性纤维化等疾病。麦芽糖转运体是一种大型 由三种不同的多肽形成的多亚单位蛋白质复合体， 由一种麦芽、一种麦芽和两种麦芽组成。申请人有 开发了一种提纯方法，可以生产毫克量的 活性麦芽糖。目前的建议是确定 生产麦芽糖转运蛋白的结晶条件 适合后续晶体结构的晶体 决心。这将需要(1)蛋白质的特性 在各种洗涤剂中，(2)均相麦芽糖转运体的生产 选择毫克量的洗涤剂；(3)溶液的研究 洗涤剂在聚乙二醇组分中的相行为 沉淀剂，(4)蛋白质的结晶实验。 使用结晶基质的选定洗涤剂通常 与水溶蛋白的结晶有关，以及(5) 使用添加剂改善结晶条件，如 两亲性、二价阳离子和其他具有高活性的洗涤剂 临界胶束浓度。将对水晶进行评估 并用不同的低温保护剂对其进行了测试。 结晶学。目标是获得高质量的晶体，并 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作用机制。这 结构应该揭示支配分子的一般原理 ABC超家族成员的机制，并提供对 与关节缺陷相关的疾病的结构基础 传送者。

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.