Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal J alpha helix that is docked on a beta-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D2O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 mu s. We observe a two-step unfolding of the J alpha helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 mu s, along with hydrogen-bond rupture of the FMN C4=O with Gln-513, motion of the beta-sheet, and an additional helical element. The second phase occurs in approximately 240 mu s. The final spectrum at 500 mu s is essentially identical to the steadystate light-minus-dark Fourier transform infrared spectrum, indicating that J alpha helix unfolding is complete on that time scale.