both could be fluorescent. However, some mutants revealed a non-planar trans form,
which may correspond to disorder or flexibility in the chromophore structure [ 26 ].
These disordered forms were found to be essentially non-fluorescent.
Quite recently, detailed quantum chemical calculations on the possible reaction
pathways in Dronpa were performed by Li and co-workers [ 140 ]. They confirmed
that a balance of proton-transfer and isomerisation steps may be important in the
photoreversible process, and considered a number of possible mechanisms. Clearly,
some detailed observations of the primary excited state processes in photoconver-
tible CPs will be critical in developing a unified picture of the mechanism(s)
operating. In particular, a correlation of excited state dynamics and time-resolved
vibrational spectroscopy will be highly informative, especially if it is accompanied
by structure determinations. Such measurements are in progress.
Since their first discovery and application in bioimaging, the range and capabilities
of the CP family have continued to grow. Recent years have seen dramatic progress
particularly in the area of photoconvertible CPs, which have already proved to be of
great utility in bioimaging. However, there is already evidence that this is by no
means the end of the CP story. In the future, CPs may contribute to life sciences as
much more than simply passive fluorescent probes. CPs with potential application in
phototherapy have already been proposed [ 141 ]. Very recently, it was demonstrated
that unnatural amino acids can be incorporated into GFP, modifying the properties
of the chromophore [ 142 , 143 ]. This exciting result suggests an entirely new range
of engineered applications. At each new step in the CP story, the advances in protein
science have been accompanied by detailed physical and chemical measurements of
the underlying mechanism. Spectroscopic and kinetic studies have in turn been
complemented by quantum chemical and molecular mechanics calculations. As
the variety and range of CPs continues to grow, so will the need for the detailed
understanding of the underlying photophysical phenomena that dynamics, spectros-
copy, theoretical calculation and molecular dynamics simulation can supply.
Acknowledgements I am grateful to EPSRC for financial support (EP/H025715), to my students
and postdoctoral fellows for their work and insights over the years, and to my collaborators for
their generous advice and assistance.
1. Shimomura O, Johnson FH (1969) Properties of bioluminescent protein aequorin. Biochem-
2. Morise H, Shimomur O, Johnson FH, Winant J (1974) Intermolecular energy-transfer in
bioluminescent system of Aequorea. Biochemistry 13:2656-2662
3. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as
a marker for gene-expression. Science 263:802-805