Structural Studies of the Multifunctional PutA Protein

多功能PutA蛋白的结构研究

• 批准号: 7009382
• 负责人: JOHN J TANNER
• 金额: $ 0.88万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-05 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7009382
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; X ray crystallography; bacterial proteins; enzyme activity; flavoproteins; gel mobility shift assay; genetic regulatory element; proline; protein metabolism; protein structure function; site directed mutagenesis; western blottings

DESCRIPTION (provided by applicant): The goal of this project is to characterize structure-function relationships for the multifunctional flavoprotein, PutA from Escherichia coli. This remarkable protein is both a transcriptional repressor of the proline utilization (put) regulon and a membrane-associated proline catabolic enzyme. The three-dimensional structural basis for the versatility of PutA is unknown. The working hypothesis whereby PutA changes its intracellular location and function is that conformational changes governed by the flavin redox state control its macromolecular associations (i.e. DNA and membrane-binding). The proposed research addresses three fundamental outstanding questions related to PutA structure and function: (1) What is the three-dimensional structure of PutA? (2) How does PutA interact with DNA? and (3) What are the conformational changes that allow PutA to function as both a DNA-binding protein and a membrane bound enzyme? The first aim of this proposal is to determine the three-dimensional structure of PutA using X-ray crystallography. Crystallization of PutA is challenging due to its large size (1320 amino acid residues) therefore a "divide and conquer" strategy will be employed in which shorter polypeptides that retain one or more of the functions of PutA will be engineered and crystallized separately. These smaller structures will then be stitched together computationally to derive a model of the full-length protein. Good progress has already been made using this approach - the 2.0 A crystal structure of a protein corresponding to the first 669 residues of PutA has been solved. The second aim is to determine the structural basis for PutA-DNA interactions by solving the crystal structures of PutA and truncated PutA proteins complexed with well-defined DNA binding sites. The third aim is to explore the conformational changes induced by proline reduction of the flavin by determining the crystal structures of PutA and truncated PutA proteins in the proline-reduced state. These studies will contribute pivotal understanding into the regulatory mechanism of PutA and timely knowledge of its structure.

描述（申请人提供）：该项目的目的是 表征多功能的结构功能关系 黄蛋蛋白，来自大肠杆菌的Puta。这种非凡的蛋白质都是 脯氨酸利用率（PUT）调节的转录阻遏物和A 膜相关的脯氨酸分解代谢酶。三维结构 Puta多功能性的基础是未知的。工作假设 puta改变其细胞内位置和功能是构象 黄素氧化还原国家控制其大分子的变化 关联（即DNA和膜结合）。拟议的研究讲话 与PUTA结构和功能有关的三个基本杰出问题： （1）PUTA的三维结构是什么？ （2）Puta如何互动 与DNA？ （3）什么是构象变化，使Puta得以实现 既是DNA结合蛋白，又充当膜结合酶？第一个 该提议的目的是确定puta的三维结构 使用X射线晶体学。 PUTA的结晶由于其挑战性 大尺寸（1320个氨基酸残基）因此“划分和征服”策略 将采用保留一个或多个的较短多肽 PUTA的功能将进行设计并分别结晶。这些较小 然后将结构缝合在一起，以得出一个模型 全长蛋白。已经使用了这个取得了良好的进步 方法-2.0与第一个相对应的蛋白质的晶体结构 已解决了669个Puta的残留物。第二个目的是确定 通过求解Puta-DNA相互作用的结构基础 Puta和截短的Puta蛋白与定义明确的DNA结合位点复合。 第三个目的是探索脯氨酸引起的构象变化 通过确定Puta和Puta的晶体结构的减少 在脯氨酸还原状态下截短的普塔蛋白。这些研究会 对PUTA的调节机制有助于关键理解和 及时了解其结构。