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Fig. 8 Molecular structure of
the red-emitting kaede
chromophore through the addition of a third ring conjugated to the original structure
(Fig. 8 ). It was shown through mutagenesis that a histidine residue adjacent to the
original chromophore is critical in forming this three ring system [ 29 ].
From the point of view of primary photochemical processes, the most intriguing
result is that the photoconversion occurs exclusively from the neutral form of the
chromophore, which has the same chemical structure (though not environment) as
in avGFP [ 124 ]. This strongly suggests a role for ESPT, which accordingly is a
feature of most of the proposed photoconversion mechanisms [ 10 , 124 , 126 ],
though not all of them [ 127 ]. Specifically, it has been proposed that an initial
ESPT step is followed by a reorganisation of the electronic structure of the anionic
form of the chromophore in its excited state resulting in the extension of the
chromophore (i.e., incorporation of the histidine residue) and a simultaneous
bond scission of the main amino acid chain. The breaking of the chain was
shown to be a feature of the photoconversion [ 29 ]. A number of further atom
transfer and electronic structure changes result in the final red-absorbing chromo-
phore. Related observations have been made on the engineered protein kikGR,
which has a number of useful properties, including being readily titratable. Intrigu-
ingly, the red form of this protein has been shown to have a different chromophore
structure compared to the kaede/EosFP, in which the third ring has a cis orientation
with respect to the newly formed double bond, showing that rotation has occurred,
which has important implications for the likely intermediate structures. Again, only
excitation of the neutral state leads to photoconversion [ 128 ].
The role of ESPT appears critical at some point in the mechanism, because the
photoconversion does not occur from the directly excited anionic chromophore
even though it is readily excited from its ground state, and can be prepared in high
yield by increasing slightly the pH. The action spectrum for photoconversion
instead follows closely the absorption of the neutral form. Unfortunately the precise
role of the ESPT is yet to be determined. One possibility is that the proton
transferred is actively involved in the chemistry. It was suggested that protonation
of the His residue via a proton wire, such as was observed in avGFP, might be an
important step [ 124 ]. However, thus far no obvious route for such a long-range
proton transfer has been established. A second possibility is that the proton itself is
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