Expression and Purification of an Archaeal Cysteine-less LOV Domain

LOV (Light Oxygen Voltage) domains are ubiquitous blue light sensors widespread in all three domains of life. They mediate signal transduction in diverse biological processes and can be useful for practical applications in optogenetics and fluorescence microscopy. The canonical mechanism of LOV domains signal transduction is based on formation of the covalent adduct between flavin chromophore and the highly conserved cysteine in the active site of the protein leading to downstream signaling cascade. However, previous studies showed that there are natural cysteine-less LOV domains enabling signal transduction via flavin photoreduction. In the absence of the conserved cysteine, photoexcitation causes flavin one electron reduction to the neutral semiquinone radical state and subsequent structural reorganizations as in the canonical photocycle. In this work, previously uncharacterized natural cysteine-less LOV domain from thermophilic haloarchaeon Halanaeroarchaeum sulfurireducens, named HsuLOV, was successfully expressed in Escherichia coli and purified in soluble form. Although HsuLOV bound a small amount of flavin when overexpressed in E. coli, the chromophore load was increased by protein incubation with flavin mononucleotide in vitro. It was demonstrated that fluorescence spectra of purified and chromophore-loaded HsuLOV are typical for LOV domains. Finally, the comparison of absorption spectra of dark- and light-adapted states of HsuLOV revealed flavin photoreduction to the neutral semiquinone radical state, thus confirming photoactivity of HsuLOV.