MOLECULAR AND CHEMICAL DESCRIPTION OF CFTR FUNCTION

CFTR 功能的分子和化学描述

• 批准号: 2518306
• 负责人: PETER L PEDERSEN
• 金额: $ 18.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518306
• 关键词: X ray crystallography; active sites; adenosine triphosphate; adenosinetriphosphatase; cell membrane; chemical binding; chemical models; chimeric proteins; chloride channels; cystic fibrosis; hydrolysis; molecular pathology; nuclear magnetic resonance spectroscopy; phosphorylation; protein purification; protein sequence; protein structure function; site directed mutagenesis; structural biology; transfection

DESCRIPTION: (From PI's description) The long range objective of this proposal is to obtain a molecular and chemical description of both the wild type CFTR protein and those mutant forms that cause cystic fibrosis. Work during the first 4 years of this project resulted in some novel, exciting and definitive progress. Both putative nucleotide domains (NBF1 and NBF2) of CFTR were shown by direct methods to bind ATP and ADP; the major disease causing mutation deltaF508 was shown to induce a marked instability and cause partial unfolding within NBF1; and microcrystals of an NBF1 fusion protein were obtained. In more recent work, NBF1 was shown to catalyze ATPase activity, be phosphorylated by protein kinase A, and associate with the membrane. In addition, 3-dimensional models of both NBF1 and NBF2 based on x-ray structures of known ATPases have been constructed for the first time. Future work will focus on relating the ATP binding and hydrolytic functions of NBF1 to a) phosphorylation capacity, b) structure, c) membrane binding, d) capacity to interact with and be modulated by NBF2, and e) mutant forms that cause cystic fibrosis. Specific aims are 5-fold and will be to: 1. Define those conditions which are optimal for supporting the ATP binding and ATP hydrolytic functions of NBF1 by carefully examining thereon both the effect of covalent phosphorylation and the effect of physiological anions and cations. 2. Identify, using site directed mutagenesis and a 3-dimensional model as a guide, those amino acids most critical for the ATP binding and ATP hydrolytic functions of NBF1, and the effect on these functions of disease causing mutations residing within this domain. 3. Elucidate the nature of the membrane binding interaction of NBF1, recently discovered in this laboratory, when the peptide segment G404-N432 preceding the domain is present. 4. Establish whether NBF2 exhibits the capacity to hydrolyze or only bind ATP, and the extent to which this domain in the absence or presence of the R domain interacts with NBF1 in a functionally important manner. 5. Vigorously continue ongoing experiments to obtain preparations of NBF1 suitable for 3-dimensional structural analysis. These studies are fundamental to understanding those structure-function relationships within both wild type CFTR and in mutant forms thereof which cause most cases of cystic fibrosis.

描述：（从PI的描述）此的远程目标 建议是获得野外的分子和化学描述 CFTR蛋白型和引起囊性纤维化的突变体形成。 工作 在该项目的前四年中，导致了一些令人兴奋的小说 和确定的进步。 两个推定的核苷酸结构域（NBF1和NBF2） 通过直接方法显示了CFTR的CFTR，以结合ATP和ADP；主要疾病 引起突变deltaf508被证明会引起明显的不稳定性和 导致NBF1内的部分展开； NBF1融合的微晶 获得蛋白质。 在最近的工作中，NBF1被证明是催化的 ATPase活性，通过蛋白激酶A磷酸化，并与 膜。 另外，基于NBF1和NBF2的3维模型 在第一个已知ATPases的X射线结构上已构建 时间。 未来的工作将集中于关联ATP结合和水解 NBF1至a）磷酸化能力的功能，b）结构，c）膜 绑定，d）与NBF2相互作用和调节的能力，E） 引起囊性纤维化的突变体形成。 具体目标是5倍，将会 到：1。定义那些最适合支持ATP的条件 通过仔细检查NBF1的结合和ATP水解功能 共价磷酸化的作用和生理的影响 阴离子和阳离子。 2。使用位点定向诱变和A 3维模型作为指导，那些对ATP最重要的氨基酸 NBF1的结合和ATP水解函数，对这些功能的影响 引起该域内突变的疾病功能。 3。 阐明NBF1的膜结合相互作用的性质，最近 在该实验室中发现的肽段G404-N432 存在域。 4。确定NBF2是否表现出 水解或仅结合ATP，以及该域中该域的程度 R域的不存在或存在与功能上的NBF1相互作用 重要的方式。 5。大力继续进行的实验以获得 NBF1的制剂适用于三维结构分析。 这些 研究对于理解那些结构功能是基础 野生型CFTR内的关系及其突变形式的关系 导致大多数囊性纤维化病例。