Biophysics of the green fluorescent protein.

It is almost certainly a truism that interpretation of the fluorescence of a protein matrix-embedded chromophore in terms of the physicochemical character of its environment requires that the tertiary structure of the protein be known to high resolution. This reality derives from the complexity of the photophysics of most fluorescent molecules, complexity that reveals the imperfections of available theory. The accuracy of these dicta is highlighted by the biophysical properties of the green fluorescent protein now being so elegantly elucidated from the application of X-ray crystallography, ultrafast optical spectroscopy, and site-specific mutagenesis. Despite the mass of recent data, however, the physicochemical basis of the green fluorescence cannot be regarded as having been fully defined, nor has the role of protein folding in chromophore formation been solved. In addition, GFP consistently yields surprises typified by the recent experiments of Vanden Bout et al. (1997) on the fluorescence of single molecules mutant (T203F and T2034) GFPs, which showed unique reversible photobleaching and were whimsically termed "blinking molecules" by Moerner (1977). Given the apparent malleability of the GFP sequence and the sensitivity of the chromophore's photophysics to a broad spectrum of physicochemical factors, it is inevitable that additional useful and intriguing biophysical properties will emerge from the study of other mutants. Although on the surface it may seem mundane, determination of the amino acid sequence and tertiary structures of the GFPs from other coelenterates is quite likely to provide very useful insights into the biophysical bases of both protein folding and the green fluorescence per se. Finally, a broader set of spectroscopic techniques need to be applied to the study of GFPs, and future fluorescence examination should include measurements of transient absorption and fluorescence emission anisotropy decays.