Strength in Numbers: Effects of Acceptor Abundance on FRET Efficiency

Abstract Fluorescence resonance energy transfer (FRET) is a strongly distance‐dependent process between a donor and an acceptor molecule, which can be used for sensitive distance measurements and characterization of molecular interactions at the nanometer level. The original mathematical description of this process, however, is only valid for the interaction of one donor with one acceptor. This criterion is not always met, especially in biological systems, where multiple structures can interact simultaneously, often making distance estimations based on transfer efficiency values error‐prone. Herein we investigate how the interaction of multiple acceptors and donors influences the transfer efficiency value in an intramolecular cellular FRET system by manipulating the fluorophore/protein ratio of the fluorophore‐conjugated antibodies. We show that the labeling ratio of the acceptor has the largest influence on measured transfer efficiency and decreasing or increasing the acceptor labeling ratio can be utilized to manipulate the FRET response of the acceptor–donor pair and therefore is a tool for optimizing sensitivity of FRET measurements.