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Abstract
Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.
Highlights
Forster resonance energy transfer (FRET) is a physical phenomenon in which excited-state energy is transferred from a donor fluorophore to near-by acceptors [1,2,3,4,5,6]
The accurate interpretation of ensemble FRET measurements requires an understanding of the underlying distribution of microscopic FRET efficiency values within the population
Monte Carlo simulations and analytical expressions were used to identify three factors that can transform a unimodal distribution of separations into a bimodal distribution of FRET efficiencies, considering the simplest case in which the donor decay in the absence of acceptor is mono-exponential
Summary
Forster resonance energy transfer (FRET) is a physical phenomenon in which excited-state energy is transferred from a donor fluorophore to near-by acceptors [1,2,3,4,5,6]. The recent discovery and optimization of genetically encoded fluorophores [9,10], GFP and its derivatives, has stimulated a renewed interest in FRET microscopy as a tool to measure protein-protein interactions within living cells (3, 4). In these experiments the average FRET efficiency, ÆEæ, is measured for a population of potential donors and acceptors. The correct interpretation of FRET measurements on such populations requires a thoughtful evaluation of the physical characteristics of the fluorophores used, of the microscopic factors that influence the efficiency of energy transfer, and an appreciation of the homogeneity of these factors [2,11]
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