Spectroelectrochemistry of the Novel PutA Flavoprotein

This project is a multi-faceted study on the regulation and structure of the flavoprotein, PutA from Escherichia coli. PutA is a remarkable multifunctional protein that autorepresses transcription of the proline utilization (put) regulon and associates peripherally with the cytoplasmic membrane where it performs the two-step oxidation of proline to glutamate. The overall goal is to determine the mechanism whereby PutA macromolecular associations (i.e. DNA and membrane binding) are regulated and to build fundamental structure-function relationships in PutA. The working hypothesis is that the flavin redox state governs PutA macromolecular associations and thus the intracellular location and function of PutA. The first two aims of this proposal are to determine the roles that the flavin redox state and substrate have in regulating putA transcription and PutA-membrane binding. The redox dependence of PutA macromolecular associations will be assessed using spectroelectrochemistry, controlled potentiometry, substrate analogs, gel-shift assays, and a liposome experimental model system. The third aim is to identify the DNA and membrane binding domains in PutA using molecular genetic techniques. Basic structure-function relationships will also be developed by correlating conformational changes in PutA with the flavin redox state and membrane binding. The final aim is to solve the three- dimensional structures of PutA and PutA functional domains using X-ray crystallography. These studies will contribute pivotal understanding into the regulatory mechanism of PutA and timely knowledge of its structure.