Light Oxygen Voltage (LOV) domains, members of the Per/Arnt/Sim (PAS) family, are blue light-responsive photosensors found in all kingdoms of life except animals (Conrad et al., 2014). They bind flavins (flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, or riboflavin, RF) as chromophores and have absorption spectra with a maximum at about 450 nm. During the photocycle, highly conserved cysteine of LOV domains forms a thioadduct with the C4a atom of the flavin, that subsequently dissociates spontaneously with recovery time values ranging from few seconds to thousands of seconds (Pudasaini et al., 2015, Zoltowski et al., 2009).

Besides being employed widely in optogenetics, LOV domains have also been developed into flavin-based fluorescent proteins (FbFPs): mutation of the conserved cysteine to a non-reactive amino acid inhibits the photocycle and enhances the fluorescence. O2-independent and relatively small in size, FbFPs are a valuable alternative to GFP-based fluorescent proteins for studying biological processes under aerobic and anaerobic conditions (Song et al., 2013). Intensive studies of LOV domains of the last two decades led to engineering of FbFP variants with increased thermal stability (Remeeva et al., 2020), covalently attached flavins (Tong et al., 2023), increased brightness (Ko et al., 2019; Liang et al., 2022), and shifted emission spectra for three-color imaging (Nikolaev et al., 2023). One of the major goals was development of photostable FbFPs, with iLOV (Chapman et al., 2008), phiLOV2.1 (Christie et al., 2012) and phiLOV3 (Babakhanova et al., 2021) being prominent examples.

One of the common sources of well-behaving proteins are thermophilic microorganisms. Accordingly, Wingen et al. tested a panel of thermophile-derived FbFPs and identified a number of promising variants (Wingen et al., 2017), including MrFbFP, which is based on a LOV domain of a histidine kinase from Meiothermus ruber, a Gram-negative, thermophilic bacterium that grows in a temperature range from 35 to 70 °C, with an optimum at 60 °C (Tindall et al., 2010). MrFbFP displayed outstanding photostability, surpassing the analogues by more than 10 times, and high thermal stability, with melting temperature of ∼ 75 °C. Yet, further functional and structural characterization of MrFbFP, or the M. ruber LOV domain, from which it was derived, is not currently available, precluding its further development. We note that MrFbFP variant studied by Wingen and colleagues contained an N-terminal elongation, which is not present in the UniProt record for a parent protein, likely disordered due to presence of a proline and two glycine residues, and might cause cross-linking via disulfide bridges due to presence of a cysteine.

Here, we aimed to study the optical and structural properties of the core LOV domain from M. ruber, MrLOV, and its extended version MrLOVe, selected based on a structural model, keeping in mind that shorter variants are preferred for cloning and tagging, but larger constructs might be more stable and suitable for studies of signal transduction. We show that the two proteins have classical PAS domain folds, both are extremely thermostable and relatively slowly recovering.