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

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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.