Tight control over gene expression is critical for normal cellular development, response to environmental signals, growth and proliferation. The broad, long-term objective of the proposed research is to understand the mechanisms by which cells use ligand-protein and protein-protein interactions to allosterically regulate gene expression. To understand allosteric gene regulation we have chosen a biochemically tractable gene regulatory system in Bacilli: control of tryptophan biosynthesis genes by the oligomeric TRAP and Anti-TRAP proteins. Specifically, this proposal focuses on characterizing the structural and dynamic basis for (1) positive regulation of the ring-shaped TRAP 11-mer protein by its allosteric activator, tryptophan, and (2) negative regulation of TRAP by the Anti-TRAP 3-/12-mer. I. We hypothesize that tryptophan binding activates TRAP by altering the protein's dynamic behavior. We will test this hypothesis by comparing the solution structures (aim 1) and dynamics and thermodynamics (aim 2) of apo and Trp-activated TRAP, as well as a constitutively active mutant, and by measuring the kinetics of TRAP activation (aim 3). We will employ NMR spectroscopy and small angle X-ray scattering (SAXS) to determine solution structure and dynamics, and calorimetric and spectroscopic methods to characterize thermodynamics and kinetics of tryptophan-mediated TRAP activation. II. We hypothesize that Anti-TRAP (AT) binds TRAP in an asymmetric fashion and that protein dynamics is critical for specific recognition of undecameric (11-mer) TRAP by trimeric/dodecameric AT. To provide insight into the mechanism of TRAP inactivation by AT, we will (aim 4) characterize the solution structure and dynamics of AT, (aim 5) study their interaction and develop a three-dimensional model of the TRAP-AT complex. Understanding the control of gene expression is a crucial step toward elucidating the molecular mechanisms of many diseases caused by improper gene regulation, such as cancer. Protein dynamics and allostery are intimately linked;therefore, mechanistic understanding of how regulation is achieved by changing protein behavior will help us to understand and ultimately control gene expression as part of a therapeutic strategy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Wehrle, Janna P
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Ohio State University
Schools of Arts and Sciences
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Ihms, Elihu C; Kleckner, Ian R; Gollnick, Paul et al. (2017) Mechanistic Models Fit to Variable Temperature Calorimetric Data Provide Insights into Cooperativity. Biophys J 112:1328-1338
Ihms, Elihu C; Foster, Mark P (2015) MESMER: minimal ensemble solutions to multiple experimental restraints. Bioinformatics 31:1951-8
Furman, Ran; Danhart, Eric M; NandyMazumdar, Monali et al. (2015) pH dependence of the stress regulator DksA. PLoS One 10:e0120746
Ihms, Elihu C; Zhou, Mowei; Zhang, Yun et al. (2014) Gene regulation by substoichiometric heterocomplex formation of undecameric TRAP and trimeric anti-TRAP. Proc Natl Acad Sci U S A 111:3442-7
Kleckner, Ian R; McElroy, Craig A; Kuzmic, Petr et al. (2013) Homotropic cooperativity from the activation pathway of the allosteric ligand-responsive regulatory trp RNA-binding attenuation protein. Biochemistry 52:8855-65
Furman, Ran; Biswas, Tapan; Danhart, Eric M et al. (2013) DksA2, a zinc-independent structural analog of the transcription factor DksA. FEBS Lett 587:614-9
Xu, Yiren; Oruganti, Sri Vidya; Gopalan, Venkat et al. (2012) Thermodynamics of coupled folding in the interaction of archaeal RNase P proteins RPP21 and RPP29. Biochemistry 51:926-35
Larue, Ross; Gupta, Kushol; Wuensch, Christiane et al. (2012) Interaction of the HIV-1 intasome with transportin 3 protein (TNPO3 or TRN-SR2). J Biol Chem 287:34044-58
Kleckner, Ian R; Foster, Mark P (2012) GUARDD: user-friendly MATLAB software for rigorous analysis of CPMG RD NMR data. J Biomol NMR 52:11-22
Kleckner, Ian R; Gollnick, Paul; Foster, Mark P (2012) Mechanisms of allosteric gene regulation by NMR quantification of microsecond-millisecond protein dynamics. J Mol Biol 415:372-81

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