Engineered Arabidopsis Blue Light Receptor LOV Domain Variants with Improved Quantum Yield, Brightness, and Thermostability

Abstract

Despite remarkable contribution of green fluorescent protein and its variants for better understanding of various biological functions, its application for anaerobic microorganisms has been limited because molecular oxygen is essential for chromophore formation. To overcome the limitation, we engineered a plant-derived light, oxygen, or voltage (LOV) domain containing flavin mononucleotide for enhanced spectral properties. The resulting LOV variants exhibited improved fluorescence intensity (20 and 70% higher for SH3 and 70% for BR1, respectively) compared to iLOV, an LOV variant isolated in a previous study, and the quantum yields of the LOV variants (0.40 for SH3 and 0.45 for BR1) were also improved relative to that of iLOV (Q = 0.37). In addition to fluorescence intensity, the identified mutations of SH3 enabled an improved thermostability of the protein. The engineered LOV variants with enhanced spectral properties could provide a valuable tool for fluorescent molecular probes under anaerobic conditions.

Experimental characterization ofin silicored-shift predicted iLOVL470T/Q489Kand iLOVV392K/F410V/A426Smutants

“…Previous efforts to improve the usability of iLOV resulted in the generation of phiLOV, an iLOV derivative which possesses a superior photostability, thus tackling one of the major drawbacks in using iLOV 23 . Very recently improved brightness of iLOV variants have been reported 13,14 . It remains to be seen whether the introduced mutations responsible for the increased photostability and brightness of new iLOV variants can be applied to improve the visualization of iLOV V392K , without interfering with its excitation and emission spectra.…”

“…So far, all experimentally obtained FbFPs share spectral features similar to GFP 12 . However, FbFPs possessed relatively weak fluorescent intensity until very recently 13,14 . Engineering a red-shifted FbFP could address this problem by shifting the excitation and emission peaks away from the cellular autofluorescence.…”

Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans

“…Earlier studies focused on the effects of mutating the conserved cysteine, which forms a covalent bond with the flavonoid cofactor during the photocycle, and some random mutations on flavin binding and photochemical reactivity [55,56]. Later studies probed the effects of mutations on other properties, particularly the absorption spectrum [47,50,57,58], photocycle lifetime [57,59], brightness of the cysteine-less variants [9,19,20,60], generation of radicals [15,61,62] and thermal stability [21][22][23]. Many of these mutations were rational, or could be rationalized after initial discovery, thus allowing one to apply the same principles to impart a different LOV domain with the desirable properties.…”

“…We expect that the effects of the proline substitutions may also be transferable to other LOV domains, since the rationales that we employed (consensus design [26][27][28], stabilization by prolines [27,28]) will hold. Interestingly, out of three LOV domain thermal stabilization studies [21][22][23], only one reported the proline substitutions [22], which were, however, not included in the most stable variant with multiple mutations. Thus, testing the effects of proline substitutions may be a complementary approach to recombination, and directed evolution to speed up the discovery of the most stable variants.…”

“…Recombination and directed evolution have been used to develop iLOV and phiLOV proteins, which were brighter and more photostable compared to their natural counterparts [19,20]. Rational engineering has been used to thermostabilize YtvA [21], while plasmid recombineering and directed evolution have been used to thermostabilize iLOV [22,23]. As an alternative to the engineering of mesophilic proteins, different LOV domains from thermophilic microorganisms have been screened for thermo-and photostability [24].…”