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 i s 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 i s 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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Metallobiochemistry Study Section (BMT)
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Ikeda, Richard A
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University of Nebraska Lincoln
Schools of Earth Sciences/Natur
United States
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Korasick, David A; Campbell, Ashley C; Christgen, Shelbi L et al. (2018) Redox Modulation of Oligomeric State in Proline Utilization A. Biophys J 114:2833-2843
Tanner, John J; Fendt, Sarah-Maria; Becker, Donald F (2018) The Proline Cycle As a Potential Cancer Therapy Target. Biochemistry 57:3433-3444
Korasick, David A; Gamage, Thameesha T; Christgen, Shelbi et al. (2017) Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis. J Biol Chem 292:9652-9665
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Liu, Li-Kai; Becker, Donald F; Tanner, John J (2017) Structure, function, and mechanism of proline utilization A (PutA). Arch Biochem Biophys 632:142-157
Moxley, Michael A; Zhang, Lu; Christgen, Shelbi et al. (2017) Identification of a Conserved Histidine As Being Critical for the Catalytic Mechanism and Functional Switching of the Multifunctional Proline Utilization A Protein. Biochemistry 56:3078-3088
Arentson, Benjamin W; Hayes, Erin L; Zhu, Weidong et al. (2016) Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA. Biosci Rep 36:
Tanner, John J (2016) Empirical power laws for the radii of gyration of protein oligomers. Acta Crystallogr D Struct Biol 72:1119-1129
Luo, Min; Gamage, Thameesha T; Arentson, Benjamin W et al. (2016) Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function. J Biol Chem 291:24065-24075

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