Irregular Excess Energy Transfer Observed with a Cerulean Donor and Multiple Venus FRET Acceptors

Abstract

Forster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred to only one of the acceptors present. The rate of energy transfer between the donor and each specific acceptor (kDA) can be measured in the absence of other acceptors, and these individual transfer rates can be used to predict the ensemble FRET efficiency. The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs were measured by systematically introducing point mutations to eliminate the chromophores of the other acceptors. We find that the amount of FRET with multiple acceptors is significantly greater than predicted by the sum of the individual transfer rates. We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption that the prediction calculation is based on is incorrect. These possibilities will be discussed.