Fluorescence Resonance Energy Transfer (FRET)

A particularly powerful fluorescence technique to probe membrane organization and lipid-protein interaction is fluorescence resonance energy transfer (FRET; Theodore Förster, 1949). The rate of energy transfer between a donor molecule, with fluorescence lifetime τ, and an acceptor molecule, separated by a distance R, is given by

where R₀ is the critical distance, which can be calculated from

where in turn κ² is the orientation factor, Φ_D is the donor quantum yield in the absence of acceptor, n is the refractive index, λ is the wavelength, I(λ) is the normalized donor emission spectrum, and ε(λ) is the acceptor molar absorption spectrum. In the latter equation, if the λ units used are nm, the calculated R0 has Å units.

If each donor senses a single acceptor in its vicinity, and the donor-acceptor distance is the same for all pairs, then FRET is easily used as a "spectroscopic ruler" to measure distances in the 1-10 nm range. In case the donor molecules are surrounded by a distribution of acceptors, the decay law becomes complex (Figure 1), and depends on the acceptor concentration and distribution.

Figure 1 - Cartoon and Equations for time-resolved FRET in bilayers, assuming both planar (cis) and interplanar (trans) transfer.

Membrane phase separation leads to partition of both donor and acceptor probes between the two phases. In general, the donor fraction and fluorescence properties will be different in the two phases, as will be the local acceptor concentrations. Analysis of time-resolved donor fluorescence in presence and absence of acceptor allows the recovery of all these parameters, and, from deviations to the theoretical expectations, information about domain size can be inferred (Figure 2).

Figure 2 - Schematic representation of FRET in phase separated membranes, in a situation where donor and acceptor partition preferably to different phases.

Regarding lipid-protein interaction, FRET is helpful in quantification of lipid-protein selectivity, determining the transverse location of the peptide/protein fluorophore, and probing the peptide/protein aggregation state, among other other important questions.

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