Fluorescence resonance energy transfer (FRET) is a distance-dependent physical process by which energy is transferred nonradiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole–dipole coupling.
Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. This method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.
The efficiency of FRET is dependent on the inverse sixth power of intermolecular separation, making it a sensitive technique for investigating a variety of biological phenomena that produce changes in molecular proximity. Technological advances in light microscopy imaging, combined with the availability of genetically encoded fluorescent proteins provide the tools necessary to obtain spatial and temporal distribution of protein associations inside living cells.
The widely used donor and acceptor fluorophores for FRET studies come from a class of autofluorescent proteins, called GFPs. The spectroscopic properties that are carefully considered in selecting GFPs as workable FRET pairs include sufficient separation in excitation spectra for selective stimulation of the donor GFP, an overlap (>30%) between the emission spectrum of the donor and the excitation spectrum of the acceptor to obtain efficient energy transfer, and reasonable separation in emission spectra between donor and acceptor GFPs to allow independent measurement of the fluorescence of each fluorophore. GFP-based FRET imaging methods have been instrumental in determining the compartmentalization and functional organization of living cells and for tracing the movement of proteins inside cells. FRET has been used to measure distance and detect molecular interactions in a number of systems and has applications in biology and chemistry.
FRET can be used to;
- measure distances between domains in a single protein and therefore to provide information about protein conformation
- detect interaction between proteins
- Applied in vivo, FRET has been used to detect the location and interactions of genes and cellular structures including integrins and membrane proteins
- used to obtain information about metabolic or signaling pathways
- used to study lipid rafts in cell membranes and to determine surface density in the membranes
- FRET and BRET are also common tools in the study of biochemical reaction kinetics and molecular motors.
- used for monitoring nanoparticle formation as well as pH dependent assembly and disassembly.
- FRET-based structural analysis is particularly well-suited for large membrane-associated protein complexes that are difficult to resolve via X-ray crystallography. Using the methods, site-specific labeling of membrane proteins with fluorescent probes suitable for FRET analysis is readily achievable
- used to measure dynamic changes in protein conformation as well as to test structural hypotheses arising from cryo-EM reconstructions and atomic structures from X-ray crystallographic experiments
When performing FRET experiments, care must be also taken to the method chosen for labeling interacting proteins. Two principal tools can be applied:
- fluorophore tagged antibodies
- recombinant fluorescent fusion proteins
The latter method essentially takes advantage of the discovery and use of spontaneously fluorescent proteins, like the green fluorescent protein (GFP). Until now, FRET has been widely used to analyze the structural characteristics of several proteins, including integrins and ion channels. More recently, this method has been applied to clarify the interaction dynamics of these classes of membrane proteins with cytosolic signaling proteins.