Quantitative Structure & Function of ABC Transporters

描述（由申请人提供） ATP 结合盒 (ABC) 超家族是最大、最常见的超家族之一。 高度保守的一类整合膜蛋白，参与 依赖 ATP 的溶质跨细胞膜运输。杰出成员 该家族中包括与多药耐药性相关的 P-糖蛋白 癌症化疗和囊性纤维化跨膜电导 调节器（CFTR），一种氯离子通道，其故障会导致囊性纤维化， 白种人中最常见的致命遗传病。的一项独特功能 CFTR 的特点是它有两个不同的核苷酸结合域 (NBD) 在 CFTR 通道的门控中发挥着关键作用。我们的理解 然而，调节机制的分子基础仍然很原始。 未解决的问题包括： 个人的功能角色是什么 NBD？哪个NBD打开通道，哪个NBD关闭通道？怎样做 两个 NBD 会相互作用吗？它们形成二聚体结构吗？如果是的话， 这种二聚体结构取决于通道的状态吗？这些是 广大生物学家感兴趣的基本问题。 将采用定量、多学科的方法来解决 CFTR 门控调节的分子机制。它是一个组合 结构建模、能量研究、膜片钳记录、生化 分析和化学合成。我们计划定量研究 NBD 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： 目标 1. 设计 CFTR 的核苷酸结合口袋以区分 NBD 的职能作用。 目标 2. 研究 NBD 复合物的异二聚体结构。 清晰理解CFTR功能的定量机制 对于 CFTR 相关疾病的未来治疗设计至关重要，例如 囊性纤维化和分泌性腹泻。方法和结果是 直接适用；到其他 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.