Structural Studies of the Multifunctional PutA Protein

多功能PutA蛋白的结构研究

• 批准号: 6888299
• 负责人: JOHN J TANNER
• 金额: $ 17.31万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6888299
• 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（由申请人提供）：该项目的目标是 表征多功能的结构-功能关系 黄素蛋白，来自大肠杆菌的 PutA。这种非凡的蛋白质既是 脯氨酸利用（put）调节子的转录抑制因子和 膜相关脯氨酸分解代谢酶。三维结构 PutA 多功能性的基础尚不清楚。工作假设 PutA改变其在细胞内的位置和功能是构象 由黄素氧化还原状态控制的变化控制其大分子 关联（即 DNA 和膜结合）。拟议的研究地址 与 PutA 结构和功能相关的三个基本悬而未决的问题： （1）PutA的三维结构是怎样的？ (2) PutA如何交互 与DNA？ (3) 哪些构象变化使得 PutA 能够 既作为 DNA 结合蛋白又作为膜结合酶发挥作用？第一个 该提案的目的是确定 PutA 的三维结构 使用X射线晶体学。 PutA 的结晶具有挑战性，因为 大尺寸（1320 个氨基酸残基）因此是“分而治之”策略 将使用保留一种或多种的较短多肽 PutA的功能将单独设计和结晶。这些较小的 然后将结构通过计算缝合在一起以导出模型 全长蛋白质。使用此方法已经取得了良好的进展 方法 - 对应于第一个的蛋白质的 2.0 A 晶体结构 PutA 的 669 个残基已得到解决。第二个目标是确定 通过求解 PutA-DNA 相互作用的晶体结构来了解 PutA-DNA 相互作用的结构基础 PutA 和截短的 PutA 蛋白与明确的 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 的晶体结构来减少黄素 脯氨酸还原状态下截短的 PutA 蛋白。这些研究将 为 PutA 的监管机制提供关键的理解， 及时了解其结构。