Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 187

Although photoconversion of DsRed may not be directly compared with that of
GFP, the reported non-linear power density dependence of photoconversion with
CW illumination in both cases may point at a similar mechanism. However, it
should be emphasised that in the nanosecond pulsed regime, power density depen-
dence is linear in the case of GFP [ 19 ] but not for DsRed [ 40 ].
Photoconversion is irreversible, as was already observed by Cubitt et al. [ 7 ].
However, after photoconversion very small (1-2%) absorption changes occur on
hours-days time scales that are only a fraction of the phototransformation changes
[ 19 ]. These occur at 293 K but not at cryogenic temperature, and are possibly
caused by slow structural relaxations of the protein environment that have small
effects on either the chromophore pKa or the cross-section [ 19 ]. In contrast, one
report claims the occurrence of significant thermal reversion with 60% recovery
of the lost absorption at 398 nm after 24 h dark incubation at 293 K [ 16 ]. This
observation of reversibility has, however, not been confirmed since. It did, how-
ever, lead to a proposal for a specific structural mechanism for photoconversion that
took the reversibility into account, which included the proposed syn-anti isomer-
isation of protonated Glu222 (Fig. 4 )[ 5 ]. It is unlikely that non-photochemical
reversal occurs after photoconversion [ 16 ]. It may be speculated that a contribution
of slow mixing of unexposed material into the probed volume may have contributed
to the measurements that were conducted in viscous samples containing glycerol
[ 16 ]. Presently, there is agreement in the literature that photoconversion of GFP is
fully irreversible.
With UV illumination, the photogenerated absorption at 483 nm is approxi-
mately twice the absorption decrease at 398 nm, which corresponds to a stoic-
hoimetric conversion (Fig. 6b ). With pulsed excitation at 390 nm several fold less
product absorption at 483 nm is observed, resulting from a significant photobleach-
ing process. Mass spectrometry analysis of fully photobleached samples after
prolonged exposure to 390 nm nanosecond pulses indicated the presence of multi-
ple fragments with reduced mass, showing several different cleavage reactions to be
responsible for the photobleaching reaction. These observations could indicate the
complete loss of the chromophore from the protein.
2.2 Mechanism of Photoconversion of GFP
Both UV and visible light transform the species with a neutral, phenolic chromo-
phore that absorbs at 398 nm (GFP A ) into an ionic species absorbing at 483 nm
(GFP R ), with a phenolate-containing chromophore [ 20 ]. Vibrational spectroscopy
provided essential information about the chromophore changes during photocon-
version. Difference FTIR measurements of the GFP R and the GFP A state confirmed
the deprotonation of the chromophore as the molecular basis of the photochromic
reaction [ 20 ] (Fig. 7 ). Prominent product bands in the mid-infrared region at
1,497 and 1,147 cm 1 are characteristic of the anionic phenolate group, which
confirmed and established the deprotonation of the chromophore as the basis of