Pii: S0962-8924(98)01434-2

Green fluorescent protein (GFP) has served as a versatile tool in cell biology for studying gene expression, as well as protein folding and trafficking (reviewed in Ref. 1). The utility of GFP in research applications derives from its intrinsic ability to generate fluorescence in live tissues, in the absence of any cofactor(s). By far the most frequent applications of GFP have involved its use as a genetically encoded tag, where GFP is fused in-frame to a protein of interest, and the resulting chimeric protein is expressed in an appropriate cellular background for examining protein function and fate. Fortunately, GFP can be targeted to most subcellular sites, making it feasible to examine biochemical processes in real time. In addition to these passive applications, GFP has also been used as an active indicator of cellular physiology. This might seem surprising given its extraordinarily rigid tin-can-like structure2,3, which would be predicted to be relatively insensitive to environmental signals. Wild-type GFP protein has not been particularly useful as a sensor, but several GFP mutants with distinct spectral qualities have been used as environmental sensors in various situations – for example, in monitoring local pH4. The most powerful method of constructing GFP sensors has been to exploit fluorescence resonance energy transfer (FRET) between two GFP molecules or between one GFP molecule and a secondary fluorophore. FRET is a phenomenon that occurs when two fluorophores are in sufficient proximity (,100 Å) and an appropriate relative orientation such that an excited fluorophore (donor) can transfer its energy to a second, longer-wavelength fluorophore (acceptor) in a nonradiative manner. Thus, excitation of the donor can produce light emission from the acceptor, with attendant loss of emission from the donor. Because FRET is a nondestructive spectroscopic method for measuring molecular interactions, it can be done in living cells, as represented by the ratio between donor and acceptor emission intensities.