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

有趣的黄素蛋白PutA在大肠杆菌中具有两种相互排斥的功能。 首先，在细胞质中，PutA自动抑制脯氨酸利用（put）调节子的putA和putP（脯氨酸转运蛋白）基因的转录。 其次，PutA与细胞质膜外周结合，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从转录调节因子转换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大分子缔合，从而控制PutA的细胞内位置和功能。 本项目的目的是建立黄素氧化还原状态和PutA的大分子缔合之间的关系。 前两个目的是研究黄素氧化还原状态对PutA和PutA-膜相互作用的转录调控功能的影响。 最终的目的是将PutA的构象变化与黄素减少相关联，并建立主要的结构-功能关系。 实验方法将联合收割机光谱电化学和生物物理方法相结合，为PutA的调节提供独特而重要的见解。 本项目将明确黄素氧化还原状态对PutA功能的影响，并对多功能PutA蛋白的调控机制提供关键性的理解。 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分校建立一个跨学科的生物化学学位课程。 实现这一目标的第一步是增加生物化学课程与先进的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。 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.