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Characterization of flavin binding in oxygen‐independent fluorescent reporters
Author(s) -
Anderson Nolan T.,
Weyant Kevin B.,
Mukherjee Arnab
Publication year - 2020
Publication title -
aiche journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.958
H-Index - 167
eISSN - 1547-5905
pISSN - 0001-1541
DOI - 10.1002/aic.17083
Subject(s) - fluorescence , oxygen , characterization (materials science) , chemistry , flavin group , biological system , biophysics , biochemistry , nanotechnology , materials science , biology , organic chemistry , physics , optics , enzyme
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.