The photophysics of LOV-based fluorescent proteins — new tools for cell biology

Abstract

In this study photophysical characteristics of LOV-based fluorescent proteins which are essential for analytic methods as well as imaging approaches have been comparatively analyzed in detail.

Theoretical Characterization of the Flavin-Based Fluorescent Protein iLOV and its Q489K Mutant

“…Some FbFPs show a second fluorescent maximum at the longer wavelengths instead of the shoulder; 6 in contrast, only a single emission maximum at 540 nm is detected for FMN in aqueous solution, whereas the absorption spectrum shows maxima at 450 and 370 nm. 6 Interestingly, all reported FbFPs demonstrate similar spectral properties but at the same time they may significantly differ in the fluorescence quantum yield, which can be almost twice as large as that of FMN in the aqueous solution. 6 Development of FbFP variants with the redshifted excitation and emission maxima in the visible spectral range and with an increased quantum yield constitutes an important direction of studies, which can benefit from application of computational approaches complementing experimental design.…”

“…6 Interestingly, all reported FbFPs demonstrate similar spectral properties but at the same time they may significantly differ in the fluorescence quantum yield, which can be almost twice as large as that of FMN in the aqueous solution. 6 Development of FbFP variants with the redshifted excitation and emission maxima in the visible spectral range and with an increased quantum yield constitutes an important direction of studies, which can benefit from application of computational approaches complementing experimental design. 15−17 In this work, we apply modern computational methods to characterize absorption and emission properties of the iLOV protein.…”

Flavin-based fluorescent proteins: emerging paradigms in biological imaging

“…Interestingly, Crei-LOV emerged as one of the brightest known FbFPs with a quantum yield that is approximately 1.5-fold greater than the quantum yield of iLOV, measured under identical conditions. Drepper and colleagues have also exploited the natural diversity of LOV proteins to develop two new FbFPs from Dinoroseobacter shibae and from Pseudomonas putida SB1 protein (known as DsFbFP and Pp1FbFP, respectively; Figure 3d) [37]. Importantly, this work also reported a PpFbFP variant (Q116V) that exhibits a blue shifted fluorescence emission ($10 nm), which could potentially prove useful for multispectral imaging using FbFPs (Figure 3d).…”

“…In a recent and impressive study, Wingen et al addressed this issue by developing a robust platform for precise characterization and comparison of key spectral properties of existing FbFPs [37]. Similarly, in our lab, we have tackled this challenge by comprehensively characterizing biophysical and biochemical properties of FbFPs [38], in tandem with developing new FbFP variants using protein engineering.…”

“…Lower panel depicts a blue shifted FbFP (Q112V) that was engineered by introducing Q112V mutation in PpFbFP, a recently identified DsFbFP from Dinoroseobacter shibae, an iLOV variant known as miniSOG (indicated as SOG) that is typically used as a singlet oxygen generator, a photostable iLOV variant known as phiLOV, and a recently identified algal FbFP from Vaucheria frigida (VafLOV). Reproduced with permission from [37].…”