Abstract
Flavin-binding photoreceptor proteins sense blue-light (BL) in diverse organisms and have become core elements in recent optogenetic applications. The light-oxygen-voltage (LOV) protein Vivid (VVD) from the filamentous fungus Neurospora crassa is a classic BL photoreceptor, characterized by effecting a photocycle based on light-driven formation and subsequent spontaneous decay of a flavin-cysteinyl adduct. Here we report that VVD presents alternative outcomes to light exposure that result in protein self-oxidation and, unexpectedly, rise of stability through kinetic control. Using optical absorbance and mass spectrometry we show that purified VVD develops amorphous aggregates with the presence of oxidized residues located at the cofactor binding pocket. Light exposure increases oxidative levels in VVD and specific probe analysis identifies singlet oxygen production by the flavin. These results indicate that VVD acts alternatively as a photosensitizer, inducing self-oxidative damage and subsequent aggregation. Surprisingly, BL illumination has an additional, opposite effect in VVD. We show that light-induced adduct formation establishes a stable state, delaying protein aggregation until photoadduct decay occurs. In accordance, repeated BL illumination suppresses VVD aggregation altogether. Furthermore, photoadduct formation confers VVD stability against chemical denaturation. Analysis of the aggregation kinetics and testing of stabilizers against aggregation reveal that aggregation in VVD proceeds through light-dependent kinetic control and dimer formation. These results uncover the aggregation pathway of a photosensor, where light induces a remarkable interplay between protein damage and stability.
Highlights
Flavin-binding photoreceptors mediate blue-light (BL) sensing in diverse organisms [1,2], and have received considerable attention recently as genetically-encoded fluorescent and optogenetic tools [3,4]
A clearly noticeable absorbance shift systematically presented at all wavelengths (Fig 1A), increasing with time, due to a change in turbidity usually associated with protein aggregation [24]
The production of 1O2 by LOV domains has been anticipated based on the feasibility of energy transfer from the flavin excited triplet state (TS) to O2 [28]
Summary
Flavin-binding photoreceptors mediate blue-light (BL) sensing in diverse organisms [1,2], and have received considerable attention recently as genetically-encoded fluorescent and optogenetic tools [3,4]. Light perception mechanisms in these proteins involve light absorption by the chromophore, followed by rearrangements of electronic structure at and near the flavin, resulting in a conformational change that drives the photoreceptor to its signaling state [5]. Oxidative damage and protein stability in Vivid collection and analysis, decision to publish, or preparation of the manuscript
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