Quantitative Structure & Function of ABC Transporters

描述(由申请人提供) 三磷酸腺苷结合盒(ABC)超家族是最大和最 高度保守的一类完整的膜蛋白，参与了 依赖于ATP的溶质跨细胞膜的运输。杰出成员 该家族包括与多药耐药相关的P-糖蛋白 肿瘤化疗与囊性纤维化的跨膜电导 调节器(CFTR)，一种氯通道，其故障会导致囊性纤维化， 高加索人最常见的致命遗传病。的一个独特功能 Cftr是它有两个不同的核苷酸结合域(NBD)，它们扮演着 在CFTR通道选通中的关键作用。我们对这一现象的理解 然而，调控机制的分子基础仍然是原始的。 悬而未决的问题包括：个人的职能作用是什么 NBD？哪个NBD打开通道，哪个NBD关闭通道？你是如何 两个NBD相互作用？它们形成二聚体结构吗？如果是， 这种二聚体结构依赖于通道的状态吗？这些是 引起广泛生物学家兴趣的基本问题。 将采用量化、多学科的方法来解决 调控CFTR门控的分子机制。它是一种组合 结构建模、能量研究、膜片钳记录、生化 化验和化学合成。我们计划对该事件进行定量调查 NBDS在CFTR通道门控中的功能作用两个平行的目标 在这个项目中有： 目的1.设计cftr的核苷酸结合口袋，以区分 NBD的职能作用。 目的2.研究NBD络合物的异二聚体结构。 认清CFTRs作用的量化机制 对于未来CFTR相关疾病的治疗设计是必不可少的，例如 囊性纤维化和分泌性腹泻。方法和结果是 直接适用于其他ABC的定量结构-功能研究 转运蛋白。

DESCRIPTION (provided by applicant) The ATP-binding cassette (ABC) superfamily is one of the largest and most highly conserved class of integral membrane proteins and is involved in the ATP-dependent transport of solutes across cellular membranes. Prominent members in this family include P-glycoprotein linked to multidrug resistance to chemotherapy for cancers and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a chloride channel whose malfunction causes cystic fibrosis, the most common lethal genetic disease in Caucasians. One unique feature of CFTR is that it has two distinct nucleotide binding domains (NBDs) that play critical roles in the gating of CFTR channels. Our understanding of the molecular basis of the regulation mechanisms, however, remains primitive. Unresolved questions include: What is the functional role of the individual NBD? Which NBD opens the channel, and which NBD closes the channel? How do the two NBDs interact with each other? Do they form a dimeric structure? If yes, does this dimeric structure depend on the state of the channel? These are the fundamental questions that interest a broad spectrum of biologists. A quantitative, multi-disciplinary approach will be used to tackle the molecular mechanisms whereby CFTR gating is regulated. It is a combination of structural modeling, energetic studies, patch-clamp recordings, biochemical assays and chemical synthesis. We plan to investigate quantitatively the functional roles of NBDs in the gating of CFTR channels. The two parallel aims in this project are: Aim 1. To engineer the nucleotide-binding pockets of CFTR to distinguish the functional roles of the NBDs. Aim 2. To investigate the hetero-dimeric structure of the NBD complex. A clear understanding of the quantitative mechanisms of the functions of CFTR is essential to future therapeutic design for CFTR-related diseases such as cystic fibrosis and secretory diarrhea. The methods as well as the results are directly applicable; to quantitative structure-function studies on other ABC transporter proteins.