Structural Studies of the Multifunctional PutA Protein

描述(由申请人提供)：本项目的目标是 表征多功能的结构-功能关系 从大肠杆菌中提取的黄素蛋白。这种非凡的蛋白质既是一种 脯氨酸利用(Put)调节子的转录抑制因子和a 膜相关的脯氨酸分解代谢酶。立体结构 普塔的多功能性的基础是未知的。工作假说，由此 Puta改变了它在细胞内的位置和功能是构象 黄素氧化还原态控制其大分子的变化 结合(即DNA和膜结合)。建议的研究内容包括 与Puta结构和功能相关的三个基本悬而未决的问题： (1)Puta的三维结构是什么？(2)Puta是如何相互作用的 用DNA吗？以及(3)什么构象变化允许Puta 既是DNA结合蛋白又是膜结合酶？第一 这项建议的目的是确定Puta的三维结构 使用X射线结晶学。普塔的结晶是具有挑战性的，因为它 大体积(1320个氨基酸残基)因此是一个“分而治之”的策略 将采用保留一个或多个 PUTA的功能将分别进行工程设计和结晶。这些更小的 然后，通过计算将结构缝合在一起，以推导出 全长蛋白质。利用这一技术已经取得了良好的进展 方法-蛋白质的2.0A晶体结构对应于第一个 解决了普塔残留量669个。第二个目标是确定 PUTA与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 通过测定Puta和Puta的晶体结构来还原黄素 脯氨酸还原状态下的截短Puta蛋白。这些研究将 有助于对PUTA和PUTA的监管机制的关键理解 及时了解其结构。

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.