Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells

Defining the repertoire of interactions that a particular protein can undergo is crucial for understanding its function and regulation. Characterizing where and when these interactions occur is a major goal of cell biology. Although biochemical approaches (e.g., immunoprecipitation, pull-down assays, and cross-linking) are indispensable for identifying protein-protein interactions, they do not provide spatial and temporal information in the context of an intact cell. Conversely, immunofluorescence localization or genetically encoded fluorescent tags such as green fluorescent proteins (GFP) can provide spatial information regarding an individual protein, but little insight into its interacting partners. While co-localization of two proteins (e.g., in the same organelle) is a prerequisite for their interaction, an interaction cannot be concluded just because two proteins are co-localized by fluorescence microscopy. Clearly, a combination of fluorescence-based visualization of proteins (in their cellular context) that simultaneously provides subnanometer resolution of their proximities (i.e., whether they can physically interact) is highly desirable in nearly all areas of cell biology. For this reason, numerous approaches have been developed to meet these demands. Because a protein’s localization is one of its most basic features, there are an enormous number of reagents to visualize individual proteins by fluorescence microscopy. These include an ever-growing collection of fluorescent protein–tagged constructs as well as highaffinity mono-specific antibodies suitable for immunofluorescence. Given the wide range of color variants of both fluorescent proteins and fluorescent dyes, visualizing two or more proteins simultaneously is now routine. To convert this basic methodology to additionally report on close (subnanometer) proximities of the fluorescently marked proteins, one needs to employ fluorescence resonance energy transfer (FRET). In essence, measurement of FRET between two appropriately labeled proteins containing fluorophores with suitable properties can be used to infer the spatial and temporal characteristics of protein interactions in their native cellular environment. How does this work? FRET refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor; UNITS 4.14 & 17.1). Hence, energy that is captured by the donor upon its excitation is transferred to the acceptor. This results in the donor failing to emit a photon, while the acceptor emits a photon at its characteristic wavelength (despite the fact it was not directly excited). Although a wide variety of parameters influences the probability of FRET (see Matyus, 1992; Clegg, 1995; Wouters et al., 2001 for detailed discussions), the most important are the distance separating the donor and acceptor, and their respective fluorescence spectra. Multiple experimental methods and instruments exist for measuring FRET (Jares-Erijman and Jovin, 2003). Selecting the appropriate method and instrumentation can be daunting, even for experienced fluorescence microscopists. Each of the techniques has particular advantages and disadvantages, and the appropriateness of a technique depends on the nature of the hypothesis being tested. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor-photobleaching FRET technique (also see UNIT 17.1). In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. If the donor fluorescence now gets brighter, one can infer that it had been in sufficiently close proximity to the acceptor to undergo FRET. The extent of increase is a quantitative and direct measure of FRET efficiency. As described below, acceptor photobleaching represents a robust technique that can be exploited to detect changes in the composition and organization of subunit proteins within a multiprotein complex and even to gain insight into relative stoichiometries of proteins within the complex. To obtain high-quality FRET data, care must be taken to select appropriate controls, maximize the signal-to-noise ratio, and