A new thermostable fluorescent protein is shown to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.

Protein-fragment complementation assays are used ubiquitously for probing protein–protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from Chloroflexus aggregans, can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aβ-Bβ, Eα-Fα, and Hβ-Iβ, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent in vivo in E. coli and human cells and have low background fluorescence. Our results enable probing protein–protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools.

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Light‐oxygen‐voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore‐proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue‐shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C‐terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color‐shifted flavin based fluorescent proteins.

Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family.