The primary photochemistry is similar among the flavin-bound sensory domains of LOV (Light-oxygen-voltage) photoreceptors, where upon blue-light illumination a covalent adduct is formed on the microseconds time scale between the flavin chromophore and a strictly conserved cysteine residue. In contrast, the adduct-state decay kinetics vary from seconds to days or longer. The molecular basis for this variation among structurally conserved LOV domains is not fully understood. Here we selected PpSB2-LOV, a fast cycling (τ 3.5 min, 20°C) short LOV protein from Pseudomonas putida that shares 67% sequence identity with a slow-cycling (τ 2467 min, 20°C) homologous protein PpSB1-LOV. Based on the crystal structure of the PpSB2-LOV in the dark state reported here, we used a comparative approach, in which we combined structure and sequence information with molecular dynamics (MD) simulations to address the mechanistic basis for the vastly different adduct-state lifetimes in the two homologous proteins. MD simulations pointed towards dynamically distinct structural region, which were subsequently targeted by site-directed mutagenesis of PpSB2-LOV, where we introduced single- and multi-site substitutions exchanging them with the corresponding residues from PpSB1-LOV. Collectively, the data presented identifies key amino acids on the Aβ-Bβ, Eα-Fα loops, and the Fα helix, such as E27 and I66, that play a decisive role in determining the adduct-lifetime. Our results additionally suggest a correlation between the solvent-accessibility of the chromophore pocket and adduct-state lifetime. The presented results add to our understanding of LOV signaling, and will have important implications in tuning the signaling behavior (on/off kinetics) of LOV-based optogenetic tools.
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