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Mechanism-based tuning of a LOV domain photoreceptor

Abstract

Phototropin-like LOV domains form a cysteinyl-flavin adduct in response to blue light but show considerable variation in output signal and the lifetime of the photo-adduct signaling state. Mechanistic studies of the slow-cycling fungal LOV photoreceptor Vivid (VVD) reveal the importance of reactive cysteine conformation, flavin electronic environment and solvent accessibility for adduct scission and thermal reversion. Proton inventory, pH effects, base catalysis and structural studies implicate flavin N5 deprotonation as rate-determining for recovery. Substitutions of active site residues Ile74, Ile85, Met135 and Met165 alter photoadduct lifetimes by over four orders of magnitude in VVD, and similar changes in other LOV proteins show analogous effects. Adduct state decay rates also correlate with changes in conformational and oligomeric properties of the protein necessary for signaling. These findings link natural sequence variation of LOV domains to function and provide a means to design broadly reactive light-sensitive probes.

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Figure 1: The VVD photosensor.
Figure 2: VVD kinetics.
Figure 3: LOV domain residue conservation.
Figure 4: Proton inventory and base catalysis.
Figure 5: Crystal structures of VVD variants.

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Acknowledgements

The authors thank A. Vaidya for help with kinetic studies, K. Gardner (University of Texas Southwestern) for supplying the AsLOV2 expression clone, J. Widom for help with mutagenesis and protein expression, and the Cornell High Energy Synchrotron for access to data collection facilities. This work was supported by US National Institutes of Health grant R01- GM079679.

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B.D.Z., B.V. and B.R.C. designed experiments; B.D.Z. and B.V. carried out experiments; B.D.Z., B.V. and B.R.C. analyzed data; B.D.Z. and B.R.C. wrote the manuscript.

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Correspondence to Brian R Crane.

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Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 2382 kb)

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Zoltowski, B., Vaccaro, B. & Crane, B. Mechanism-based tuning of a LOV domain photoreceptor. Nat Chem Biol 5, 827–834 (2009). https://doi.org/10.1038/nchembio.210

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