them. Since FRET is strongly distance dependent on the nanometer scale, it is
widely used to observe protein-protein interactions [ 14 ].
Thus, pH sensitivity and ESPT are photophysical phenomena exhibited by the
original avGFP, to which may be added spectral tuning and FRET when GFP
mutants are considered. However, the chromoproteins (CPs) now being discovered
in a variety of marine organisms exhibit a still wider range of photophysical
phenomena, all of which can in turn be modified by mutagenesis. Many CPs have
a spectrum which is red-shifted compared to avGFP, because the original chromo-
phore has undergone a further post-translational reaction leading to an extension of
the pi delocalisation [ 10 ]. Some of these new CPs absorb strongly in the visible
region but exhibit such fast radiationless decay as to be considered effectively non-
fluorescent (hence CP is a preferred designation to FP; it is worth noting that CP has
also been applied to coloured proteins which bind their chromophore non-cova-
lently, it is used here as it is firmly embedded in the literature and little confusion
arises, even though autochromic protein might be the more appropriate nomencla-
ture) [ 22 ]. The mechanism is fast internal conversion (IC) and may be associated
with non-planar forms of the chromophore [ 23 ]. In some CPs, the chromophore
exists in either trans or cis forms, and under irradiation isomerisation between them
may be observed [ 24 - 26 ]. Other CPs have been shown to exhibit photochemistry
leading to large spectral shifts and changes in quantum yield, a phenomenon that
can be used in 'optical highlighting' a specific protein population in vivo [ 27 - 29 ].
Finally some proteins can be photochemically and reversibly switched between
emissive and non-emissive states, a phenomenon that forms the basis of a novel
sub-diffraction-limited microscopy method currently yielding unprecedented 3D
spatial resolution in bioimaging [ 30 - 32 ].
Thus, CPs add isomerisation, photochemistry and reversible photochromism to
the photophysical phenomena already noted in avGFP. These new phenomena have
already stimulated a number of new applications in life sciences. In this chapter, the
primary photophysics of CPs will be reviewed, with a focus on excited state
dynamics. First, the main time-resolved spectroscopic methods will be introduced.
Next, the medium and substituent sensitivity of the GFP chromophore (HBDI), the
mechanism of its fast radiationless decay and the relevance of this to photo-induced
cis - trans isomerisation will be addressed. After that the ESPT reaction in avGFP
and some of its mutants will be described. Finally, the mechanism of isomerisation
and photochemistry in CPs will be discussed in relation to the CP photochromism
described elsewhere in this volume.
2 Experimental Methods
2.1 Ultrafast Fluorescence Up-Conversion
Time-resolved fluorescence has been critical to understanding the photophysics
of fluorescent proteins and is central to their application in fluorescence lifetime