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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Oct 13, 2020
- Source ID
- 10.1002/aic.17083
Entities
People
- Arnab Mukherjee
- Kevin B. Weyant
- Nolan T. Anderson
Organizations
- Cornell University
- National Institute of General Medical Sciences
- United States Department of Defense
- University of California
- Yusuf Hamied Department of Chemistry