Figure 1

Fluorescence resonance energy transfer (FRET) is an analytical technique that is useful for investigating processes that involve a change in molecular proximity, such as the assembly of protein complexes, immunoassays and the distribution and transport of lipids.FRET relies on the distance-dependent interaction between two chromophores within the system under investigation. Specifically, this interaction involves the transfer of energy from an electronically excited donor molecule to an acceptor molecule without the emission of a photon. Usually, the donor and acceptor are different entities. A key aspect of FRET is the Förster radius, which relates interchromophore distance and spectroscopic properties for any given donor-acceptor pair. It describes the separation (in å) at which a 50% transfer of excitation energy occurs. It is important to select a donor-acceptor pair that possesses a Förster radius comparable to the spatial arrangements in the biological system being studied. For FRET to occur, there are a number of spatial and spectral criteria which must be fulfilled. The donor-acceptor pair must be in close proximity (typically 10-100 å) and their transition dipole orientations must be approximately parallel. The emission spectrum of the donor and the absorption spectrum of the acceptor must overlap to some extent. With different donor and acceptor chromophores, FRET can be detected in two ways: probably the more common method involves the measurement of donor quenching, but the appearance or enhancement of acceptor fluorescence is also useful. The latter can sometimes be complicated by the fact that many acceptors absorb some of the incident light used to excite the donor and hence exhibit a degree of fluorescence even before energy transfer occurs. In cases where the donor and acceptor are the same, FRET is monitored by fluorescence depolarization. For some time, donor and acceptor moieties have been introduced into biological systems using organic chemical techniques such as covalent bond formation with synthetic fluorescent dyes. A typical donor-acceptor pair of this type is fluorescein (donor) and tetramethylrhodamine (acceptor). The natural incidence of donors and acceptors is somewhat limited but recent years have seen the introduction of a family of molecules called green fluorescent proteins (GFPs). In the approach described by Allen et al., two GFPs - enhanced cyan fluorescent protein, ECFP, the donor (D) and enhanced yellow fluorescent protein, EYFP, the acceptor (A) - have been used, bound together via calmodulin and a calmodulin-binding peptide, M13, to form a Ca²⁺ sensor referred to as a 'cameleon'. The figure illustrates how the binding of calcium to the calmodulin domain causes conformational changes that serve to bring the ECFP donor into closer proximity to the EYFP acceptor, facilitating fluorescence resonance energy transfer. This can be monitored by the detection of enhanced emission at 535 nm. The process has the added advantage of being fully reversible, allowing ratiometric measurement of cytoplasmic free Ca²⁺.