Fig. 10 Proposed mechanism for light-induced decarboxylation of Glu 222. R1 and R2 corre-
spond to residues Phe 64 and Val 68. Asterisk denotes the short-lived (t ¼
3.6 and 12.0 ps 9 )
excited state of the neutral, phenolic chromophore. The actual distances between the
group of Glu 222 and the phenolic- and imidazolidinone rings are 3.7 and 3.8 ˚ , respectively.
ACH 2 ·radical intermediate state is expected from the “Kolbe” mechanism [ 43 , 44 ]. Recombina-
tion of this electron deficient carbon radical after decarboxylation may involve direct transfer of an
electron in addition to transfer of a proton from the chromophore, or alternatively transfer of a
hydrogen radical. Reproduced with permission [ 19 ]
First, decarboxylation of Glu 222 takes place (Fig. 10 ). This is followed by
structural rearrangements of nearby amino acids Thr 203 and His 148 (Fig. 9 ).
These thermal relaxation reactions blue-shift the absorption maximum [ 19 ].
Heating the stable photoproduct obtained at 92 to 293 K produced a photoproduct
indistinguishable from that produced at 293 K. A slow transformation was already
observed in real time (minutes) at 182 K. Re-cooling the product to 92 K, or cooling
the photoproduct obtained at 293 to 92 K, produced a blue-shifted species absorbing
at 452 and 477 nm. This indicates that at higher temperatures, a thermal relaxation
process, such as a structural reorganisation, follows the primary photoconversion
process, producing GFP R with a more strongly hydrogen bonded phenolate chromo-
phore as shown by the X-ray structure [ 19 ]. Structural changes associated with
thermal transitions were revealed by X-ray crystallographic structure determination
of intermediates after photoconversion of crystals of wild-type GFP [ 26 ]. In addi-
tion, spectroscopic changes in the visible and infrared region are observed, which
has led to the identification of discrete intermediates in this pathway. The naming for
the intermediates follows GFP A (+h
(293K), for “Lumi”, “Meta” and “Relaxed”. These intermediates are cryo-trapped,
or accumulated, at the temperatures indicated. Detailed discussion of the spectral
and structural characteristics of the intermediates [ 26 ] is beyond the scope of this
review. In summary, structural changes of individual amino acids and solvent
molecules occur in the discrete intermediates, and the available vibrational spec-
troscopy has led to assignment of modes to individual amino acids such as Gln69.
GFP L (100K)
GFP M (200K)
O mode is observed at 1,696 cm 1 in the low-temperature product GFP L ,
signalling structural perturbation of the amino acid side chain [ 26 ]. A key observa-
tion from ultrafast infrared spectroscopy was that the 1,696 cm 1 mode belonging to
Gln69 perturbation is also observed, but reversibly, in the fluorescence photocycle.
Interestingly, in H 2 O the transient is developed within 1 ps, which demonstrates one
of the most rapid amino acid structural reactions documented.
Small molecule activation and migration in proteins is an important topic, which
has, for example, been addressed in detail in myoglobin [ 45 - 47 ] The photogenerated