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Estimation of the available free energy in a LOV2-Jα photoswitch

Abstract

Protein photosensors are versatile tools for studying ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins—plant serine/threonine kinases that are activated by blue light via conserved LOV (light, oxygen and voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in a light-dependent fashion with an adjacent α-helix (Jα) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain–Jα-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by 3.8 kcal mol−1, thus quantifying the energy available through LOV-Jα for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-Jα interaction.

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Figure 1: Schematic representations of phototropin domain organization and photoswitching mechanism.
Figure 2: Conformational exchange dynamics at the LOV-Jα interface as detected by relaxation dispersion measurements.
Figure 3: 15N relaxation dispersion analyses of LOV2-Jα constructs containing V529A, V529E and V529 point mutations.
Figure 4: The Jα-helix is unfolded in the high-energy conformation of dark state LOV2-Jα.
Figure 5: The Jα-helix is largely unfolded in the lit state.

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Acknowledgements

Research was supported by grants from the Welch Foundation to M.K.R. (I–1544) and K.H.G. (I–1424) and by a grant from the Texas Higher Education Coordinating Board Advanced Technology Program (010019-0124-2003). We thank L.E. Kay (University of Toronto) for providing the pulse sequences and software for data acquisition and analyses, D.M. Korzhnev (University of Toronto) for help with experimental setup and data fitting and D. Kern (Brandeis University) for assistance with improved data acquisition methods. We also thank all members of the Rosen and Gardner labs for helpful discussions.

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X.Y., M.K.R. and K.H.G. designed experiments, analyzed data and wrote the paper, and X.Y. performed experiments.

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Correspondence to Kevin H Gardner.

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Yao, X., Rosen, M. & Gardner, K. Estimation of the available free energy in a LOV2-Jα photoswitch. Nat Chem Biol 4, 491–497 (2008). https://doi.org/10.1038/nchembio.99

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