CAREER: Spectroelectrochemical Studies of the Novel PutA Flavoprotein and its Macromolecular Associations

[01:16 . 64]有趣的黄蛋白PutA在大肠杆菌中执行两种相互排斥的功能。首先，在细胞质中，PutA自抑制脯氨酸利用（put）调控的PutA和putP（脯氨酸转运体）基因的转录。其次，PutA与细胞质膜周围结合，在那里它执行脯氨酸到谷氨酸的两步氧化。本研究的总体目标是确定PutA从转录调节因子转变为膜相关脯氨酸脱氢酶的机制。工作假设是黄素氧化还原状态控制PutA大分子结合，从而控制PutA在细胞内的位置和功能。本项目的目的是建立黄素氧化还原状态与PutA大分子结合之间的关系。前两个目的是研究黄素氧化还原状态对PutA转录调控功能和PutA-膜相互作用的影响。最终目的是将PutA的构象变化与黄素还原联系起来，并建立初步的结构-功能关系。实验方法将结合光谱电化学和生物物理方法，为PutA的调控提供独特而关键的见解。该项目将明确黄素氧化还原状态对PutA功能的影响，为了解多功能PutA蛋白的调控机制提供关键的理解。对具有多种功能的蛋白质（如PutA）的研究很重要，因为它们将提供关于特定结构支架如何用于不同目的的知识。总体教育目标是在密苏里大学圣路易斯市分校建立一个跨学科的生物化学学位课程。路易。实现这一目标的第一步是增加生物化学课程的高级课程和新的实验室技术，并扩大本科生和研究生的研究机会。对PutA的研究将促进将研究发现和应用纳入课程。

0091664BeckerThe intriguing flavoprotein, PutA, performs two mutually exclusive functions in Escherichia coli. First, in the cytoplasm, PutA autorepresses transcription of the putA and putP (a proline transporter) genes of the proline utilization (put) regulon. Secondly, PutA associates peripherally with the cytoplasmic membrane where it performs the two-step oxidation of proline to glutamate. The overall goal of this research is to define the mechanism by which PutA switches from a transcriptional regulator to a membrane-associated proline dehydrogenase. The working hypothesis is that the flavin redox state governs PutA macromolecular associations and thus the intracellular location and function of PutA. The objective of this project is to establish the relationship between the flavin redox state and the macromolecular associations of PutA. The first two aims are to investigate the influence of the flavin redox state on the transcriptional regulatory function of PutA and PutA-membrane interactions. The final aim is to correlate conformational changes in PutA with flavin reduction and establish primary structure-function relationships. Experimental approaches will combine spectroelectrochemistry and biophysical methods to provide unique and critical insights into the regulation of PutA. This project will clearly define the influence of the flavin redox state on PutA functions and contribute pivotal understanding into the regulatory mechanism of the multifunctional PutA protein. Studies of proteins that have multiple functions such as PutA are important because they will provide knowledge of a how a particular structural scaffold is used for different purposes. The overall educational goal is to establish an interdisciplinary biochemistry degree program at the University of Missouri-St. Louis. The first step toward this goal is to augment biochemistry curriculum with advanced courses and new laboratory techniques and to broaden research opportunities for undergraduate and graduate students. The study of PutA will promote incorporation of research discoveries and applications into the curriculum.