Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters

Abstract

Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)–namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4–11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10–40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.

LOV-based reporters for fluorescence imaging

“…The requirement for FMN does not restrict its utility as a fluorescent reporter, as this appears to be in plentiful supply in bacterial, plant and human cells [16 ] nor limit their targeting to subcellular compartments [16 ,17]. Moreover, the fast maturation of LOV fluorescence (with minutes) constitutes one advantage over GFP as a noninvasive real-time reporter of transcriptional activities [18]. However, it is worth noting that LOV-based FPs engineered thus far exhibit fluorescence quantum yields (Q F ) that are significantly lower (0.2-0.4; Table 1) to that of GFP (0.6) [2], which could impact their utility for certain reporter applications.…”

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

“…These cyan-green fluorescing proteins bind flavin mononucleotide (FMN) as the chromophore and are either derived from bacterial photoreceptors (FMN-binding fluorescent proteins; FbFP 8 ) or are derivatives of the Arabidopsis thaliana phototropin 2 LOV2 domain (iLOV, miniSOG; 9,12 ). In contrast to all members of the GFP family, this novel class of LOV-based FPs develops the corresponding fluorescence signal under both, aerobic and anaerobic conditions 8,13,14 . This unique property renders them valuable for in vivo applications where molecular oxygen is limited; such as the analysis of microbial pathogenesis, hypoxia induced inflammatory processes, tumor pathophysiology and microbial fermentation as well as for the monitoring and optimization of bioremediation and bacterial production processes (e.g.…”

“…For example, the use of iLOV as alternative fluorescent tag enabled advanced studies of the localization, movement and dynamics of target proteins and viruses 9,10,31,32 . Furthermore, in vivo and in vitro studies demonstrated that, in contrast to GFP-like proteins, LOV-based reporter proteins rapidly gain their fluorescence-active conformation because of their fast folding kinetics and the spontaneous incorporation of the chromophore 14,33 , thereby enabling their application as real-time reporters. Their rapid assembly as well as the robustness of the fluorescence signal have rendered LOV-based FPs valuable quantitative reporters for biotechnological approaches [33][34][35][36][37] .…”

Flavin-based fluorescent proteins: emerging paradigms in biological imaging

“…In some cases, we have observed that overexpression of FbFPs may lead to varying degrees of metabolic burden and a concomitant reduction in cellular growth rate [38]. Based on these results, we conjecture that targeted mutagenesis of FMN-binding pocket amino acids or 'insoluble' hydrophobic patches in FbFPs could be particularly useful for optimizing FbFPs for improved intracellular expression and brighter fluorescence.…”

“…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. Strikingly, our results suggest that FbFPs (in particular iLOV and CreiLOV) exhibit multiple advantages as fluorescent reporter probes, including an overall 20 Analytical biotechnology ( Figure 2 Legend Continued) as miniSOG expressed in the mitochondria as a cytochrome C fusion and imaged via fluorescent microscopy.…”