Characterization of flavin binding in oxygen‐independent fluorescent reporters

Abstract

Fluorescent proteins based on light, oxygen, and voltage (LOV) sensing photoreceptors are among the few reporter gene technologies available for studying living systems in oxygen-free environments that render reporters based on the green fluorescent protein nonfluorescent. LOV reporters develop fluorescence by binding flavin mononucleotide (FMN), which they endogenously obtain from cells. As FMN is essential to cell physiology as well as for determining fluorescence in LOV proteins, it is important to be able to study and characterize flavin binding in LOV reporters. To this end, we report a method for reversibly separating FMN from two commonly used LOV reporters to prepare stable and soluble apoproteins. Using fluorescence titration, we measured the equilibrium dissociation constant for binding with all three cellular flavins: FMN, flavin adenine dinucleotide, and riboflavin. Finally, we exploit the riboflavin affinity of apo LOV reporters, identified in this work, to develop a fluorescence turn-on biosensor for vitamin B2.

Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species

“…Dissociation constants of YtvA-LOV from B. subtilis determined using ITC experiments were K d = 0.351, 0.715 and 0.224 µM [10]. Dissociation constants of EcFbFP (variant of YtvA codon-optimized for E. coli) determined using fluorescence titration are K d = 0.76, 0.18 and 0.13 µM and of iLOV (derived from A. thaliana protein) are K d = 0.89, 0.23 and 0.18 µM [9], respectively. We note that some LOV domains, such as Vivid [43] and the LOV domain of WC-1 [44], bind exclusively FAD in their native host.…”

“…LOV domains are members of the PAS (Per-Arnt-Sim) superfamily and share the common globular alpha/beta fold [8]. In the core, LOV proteins may bind different flavin chromophores (riboflavin, RF; flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) with dissociation constants around 0.1-0.9 µM [9,10]. These flavins are present in all living organisms, and consequently, heterologous expression of LOV domains usually does not require flavin supplementation or genetic introduction of their biosynthesis pathways.…”

Computational Investigation of Structural and Spectroscopic Properties of LOV-Based Proteins with Improved Fluorescence

“… 2 , 3 In this context, the so-called flavin-based fluorescent proteins (FbFPs) emerged as an alternative class of FPs. 4 − 7 FbFPs are derived from a highly conserved family of blue light photoreceptors known as light, oxygen, and voltage (LOV) sensing proteins. In nature, LOV proteins typically associate with flavin mononucleotide (FMN) to function as blue-light photoreceptors and regulate a serie of cellular processes in both bacteria (stress response and virulence) and plants (phototaxis).…”

“…Green fluorescent protein (GFP) has revolutionized the imaging of dynamic processes within living cells . However, the use of GFP as in vivo reporters is limited by some environmental and cellular factors impeding either chromophore formation or fluorescence activity. , In this context, the so-called flavin-based fluorescent proteins (FbFPs) emerged as an alternative class of FPs. − FbFPs are derived from a highly conserved family of blue light photoreceptors known as light, oxygen, and voltage (LOV) sensing proteins. In nature, LOV proteins typically associate with flavin mononucleotide (FMN) to function as blue-light photoreceptors and regulate a serie of cellular processes in both bacteria (stress response and virulence) and plants (phototaxis). , …”

Enhanced small green fluorescent proteins as a multisensing platform for biosensor development

“…Several optimized flavin-binding FPs were shown to have high pH and temperature stability thus enabling their application in a wide range of conditions both in live cells and in vitro ( Mukherjee et al, 2015 ; Wingen et al, 2017 ). More recently, the utility of LOV-based FPs was extended to the detection of heavy metal ions including copper ( Zou et al, 2020 ) and mercury ( Ravikumar et al, 2015a ) as well as flavin derivatives ( Anderson et al, 2020 ). However, compared to GFP-like FPs, the flavin-binding FPs still have room for development regarding their biophysical and biochemical characteristics, especially intracellular fluorescent brightness and photostability, which would promote their wider adaptation as biosensors ( Ozbakir et al, 2019 ).…”