Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 189

Fig. 8 Photoconversion results in a mass reduction of 44 Da. “Maximum entropy treated ESI
mass spectrometry results are for GFP A ( solid trace ) and the photoproduct GFP R ( dotted trace ).
The calculated values for the fragments Ser 2-Met 233 and Ser 2-Tyr 237 are 26,116.4 and
26,637.0 Da, respectively, taking into account a dehydration resulting from chromophore forma-
tion. A mass reduction of 43.8 and 43.9 Da, respectively, was observed for the fragments upon
photoconversion” (reproduced with permission [ 19 ])
that a new H-bonding network replaces the H-bonding network in the ground state
(Fig. 9 ). The removal of the acidic side chain in the photoproduct destabilises the
protonated chromophore. This agrees with the model for the interactions in
the GFP A ground state proposed by Brejc et al. [ 5 ]. In order to test for the possible
accumulation of a radical state, electron spin resonance (ESR) measurements were
conducted. Under conditions of continuous illumination with 254 nm light at 200
and 293 K, no photochemically produced radical products or free spin intermediates
could be detected by ESR spectroscopy, neither directly nor by spin-trapping using
the spin traps 5,5-dimethyl-1-pyrroline- N -oxide (DPMO) and 2,2,6,6,-tetramethyl-
4-piperidone (TEMP). This places a limit on the lifetime of any intermediate radical
species from the rate of optical excitation, estimated at approximately 5 s 1 , multi-
plied by the photochemical quantum yield, 0.03, which equal ~0.15 s 1 .
The experimental observations show that light-induced electron transfer leads to
oxidation of the anionic carboxylate of Glu222, which is proposed to proceed via
direct transfer to the chromophore. Both the first (S1) and second (S2) electronically
excited state are electron deficient singlet states, and the S2 state is expected to have
the largest oxidising potential (see also below). Stark spectroscopy has shown that
the imidazolidinone ring is electron deficient in the excited state of the neutral,
phenolic chromophore [ 16 , 42 ]. Assuming that the imidazolidinone ring, in particu-
lar the carbonyl (O2) oxygen atom of the phenolic chromophore, forms a strong
oxidising moiety in the excited state, a mechanism can be proposed for the light-
activated decarboxylation reaction: Upon excitation of the chromophore of GFP,