Dissecting interacting molecular populations by FRET

Abstract

COMMENTARY Fluorescence resonance energy transfer (FRET) (1), which used to be a biophysical technique strictly for specialists a few decades ago, has become a standard tool to study molecular conformation, interactions, and colocalization at the nanometer scale in cell biology and related fields (2–9). This penetration is exemplified by new editions of cell biology textbooks covering FRET among classical methods such as coimmunoprecipitation or magnetic resonance (MR) (10). The increased popularity of FRET is due to several factors including the availability of brighter, more photostable dyes, quantum dots, and color variants of fluorescent proteins, and the development of new features, add-ons and dedicated hardware and software that are available for commercial microscopes (11) and flow cytometers (12), and facilitate the smooth application of FRET techniques. In addition to the fact that FRETmeasurements can be applied to detect molecular associations (2–9,13), the FRET phenomenon can be used to improve spectral characteristics of luminescent probes to be utilized in cytometry (14,15). In this commentary we will address two concerns frequently encountered in FRET imaging: the need for being able to dissect molecular populations displaying different FRET efficiencies (16), and the surmounting of artifacts that may arise due to molecular diffusion during the FRETexperiment (17,18). FRET is the nonradiative transfer of excitation energy from an excited, fluorescent donor dye to a (not even necessarily fluorescent) acceptor in the 2–10 nm vicinity of the donor. The probability of the process is characterized by the FRET efficiency (E), which is a measure of the fraction of excitation energy quanta transferred from the donor to the acceptor. Because of the dependence of the rate of FRET on the negative 6th power of the donor–acceptor distance, FRET is also called a spectroscopic ruler (19) allowing sensitive determination of interand intramolecular distances under ideal conditions. Because of FRET, the fluorescence intensity and lifetime of the donor dye decrease, whereas its anisotropy increases (provided that the donor and the acceptor are two different dyes and there is no significant back transfer of energy from the acceptor to the donor) (20). At the same time, the fluorescence intensity of the acceptor increases (sensitized emission) and its anisotropy decreases. Different methods to evaluate the FRET efficiency take advantage of one or more of these spectroscopic changes (3). We can classify them, somewhat arbitrarily, into (a) intensitybased methods measuring donor quenching and/or sensitized emission, and (b) lifetime-based methods determining the fluorescence lifetime components directly or instead, a spectroscopic quantity related to the lifetime (such as the characteristic donor photobleaching rate in a donor photobleaching experiment). Subcellular inhomogeneity of protein–protein interactions necessitates the resolution of high-FRET and low-FRET molecular populations within the same cell. However, by using classical intensity based methods no distinction can be made between the situation of a single population of donor–acceptor pairs characterized by a single intermediate FRETefficiency value, and a mixture of high-FRET and low-FRET donor– acceptor pairs producing the same average FRET efficiency as in the first case. The reason is that in these measurements