Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors

Srinagesh V Koushik,Paul S Blank,Steven S Vogel,Vladimir Brezina

doi:10.1371/journal.pone.0008031

Abstract

BackgroundFörster 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 in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (kD→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.Methodology/Principal FindingsThe 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 FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.Conclusions/SignificanceWe conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.

Highlights

Forster resonance energy transfer (FRET) is a near-field mechanism by which energy is transferred from a donor fluorophore to an adjacent chromophore via nonradiative dipoledipole interactions [1,2,3,4,5,6]
To test the generality of the kinetic model for FRET with multiple acceptors we engineered a set of genetic constructs composed of different mixtures and arrangements of three spectral variants of Green Fluorescent Protein (FP), using Cerulean [9], Venus [10], and Amber [11] a Venus-‘‘like’’ molecule that has a point mutation preventing fluorophore formation and presumably it can’t act as a FRET acceptor
Cerulean in ACA had a lifetime of 2.9560.02 ns when measured in living cells by time correlated single photon counting (TCSPC) [12] (Fig. 1A)

Summary

Introduction

Forster resonance energy transfer (FRET) is a near-field mechanism by which energy is transferred from a donor fluorophore to an adjacent chromophore via nonradiative dipoledipole interactions [1,2,3,4,5,6]. FRET theory is applicable only to fluorophores that have very weak coupling [7], and primarily considers the interactions of a single donor-acceptor pair when they are separated by between 1–10 nm [5,6], but can be expanded to cover the situation when more than one acceptor is present if one assumes that a donor interacts with each acceptor independently. If kDRA is the rate of energy transfer from a donor to an acceptor in the presence of a single acceptor, and tD is the fluorescence lifetime of the donor fluorophore in the absence of acceptors, E, the FRET efficiency is [4,6]: E~. The rate of energy transfer between the donor and a specific acceptor (kDRA) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates

Objectives

Methods

Results

Conclusion