High-resolution structure of a naturally red-shifted LOV domain

LOV domains are widespread photosensory modules that have also found applications in fluorescence microscopy, optogenetics, and light-driven generation of reactive oxygen species. Many of these applications require stable proteins with altered spectra. Here, we report a flavin-based fluorescent protein CisFbFP derived from Chloroflexus islandicus LOV domain-containing protein. We show that CisFbFP is thermostable, and its absorption and fluorescence spectra are red-shifted for ∼6 nm, which has not been observed for other cysteine-substituted natural LOV domains. We also provide a crystallographic structure of CisFbFP at the resolution of 1.2 Å that reveals alterations in the active site due to replacement of conservative asparagine with a serine. Finally, we discuss the possible effects of presence of cis-proline in the Aβ-Bβ loop on the protein's structure and stability. The findings provide the basis for engineering and color tuning of LOV-based tools for molecular biology. •Chloroflexus islandicus genome encodes a red-shifted LOV domain.•Homologous mutation results in red shift of fluorescence spectra in YtvA but not in CagFbFP.•High-resolution crystal structure reveals altered active site and additional water molecule.•Presence of cis-proline in the Aβ-Bβ loop changes its structure in CisFbFP.