The goal of this project is to characterize structure-function relationships for the multifunctional flavoprotein, PutA from Escherichia coli. This remarkable protein is both a transcriptional repressor of the proline utilization (put) regulon and a membrane-associated proline catabolic enzyme. The three-dimensional structural basis for the versatility of PutA is unknown. The working hypothesis whereby PutA changes its intracellular location and function is that conformational changes governed by the flavin redox state control its macromolecular associations (i.e. DNA and membrane-binding). The proposed research addresses three fundamental outstanding questions related to PutA structure and function: (1) What is the three-dimensional structure of PutA? (2) How does PutA interact with DNA? and (3) What are the conformational changes that allow PutA to function as both a DNA-binding protein and a membrane bound enzyme? The first aim of this proposal is to determine the three-dimensional structure of PutA using X-ray crystallography. Crystallization of PutA is challenging due to its large size (1320 amino acid residues) therefore a """"""""divide and conquer"""""""" strategy will be employed in which shorter polypeptides that retain one or more of the functions of PutA will be engineered and crystallized separately. These smaller structures will then be stitched together computationally to derive a model of the full-length protein. Good progress has already been made using this approach - the 2.0 A crystal structure of a protein corresponding to the first 669 residues of PutA has been solved.
The second aim i s to determine the structural basis for PutA-DNA interactions by solving the crystal structures of PutA and truncated PutA proteins complexed with well-defined DNA binding sites.
The third aim i s to explore the conformational changes induced by proline reduction of the flavin by determining the crystal structures of PutA and truncated PutA proteins in the proline-reduced state. These studies will contribute pivotal understanding into the regulatory mechanism of PutA and timely knowledge of its structure.
| Korasick, David A; White, Tommi A; Chakravarthy, Srinivas et al. (2018) NAD+ promotes assembly of the active tetramer of aldehyde dehydrogenase 7A1. FEBS Lett 592:3229-3238 |
| 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 |
| Korasick, David A; Kon?itíková, Radka; Kope?ná, Martina et al. (2018) Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants. J Mol Biol : |
| 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; Tanner, John J (2018) Determination of protein oligomeric structure from small-angle X-ray scattering. Protein Sci 27:814-824 |
| Korasick, David A; Pemberton, Travis A; Arentson, Benjamin W et al. (2017) Structural Basis for the Substrate Inhibition of Proline Utilization A by Proline. Molecules 23: |
| 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 |
| Korasick, David A; Singh, Harkewal; Pemberton, Travis A et al. (2017) Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure. FEBS J 284:3029-3049 |
| 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 |
| Christensen, Emily M; Patel, Sagar M; Korasick, David A et al. (2017) Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1. J Biol Chem 292:7233-7243 |
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