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Photoconversion of the Green Fluorescent
Protein and Related Proteins
Jasper J. van Thor
Abstract This review focuses on the mechanistic details of photochromic reactions
of the green fluorescent protein (GFP) and also of its mutant derivatives and related
fluorescent proteins. A number of distinct photochromic processes have so far been
identified that have entirely different photochemical and chemical basis, which
will be reviewed. In addition to bright fluorescence, the GFP from the jellyfish
Aequorea victoria undergoes photochromic transformation with blue or UV illumi-
nation. The associated change in electronic absorption provides a spectroscopic
contrast that can be used in fluorescence microscopy application to tag and track
the movement of populations that are photoconverted. Key to the successful use of
photoconversion for such microscopy experiments is in fact the relatively low
quantum yield of the irreversible process. In the wild-type GFP, photoconversion
is triggered by light-induced electron transfer from the buried anionic carboxylate
of Glu222 to the optically excited protonated chromophore. An unstable carboxylate
radical subsequently cleaves off a CO 2 molecule in a “Kolbe” type reaction that has
been trapped in a partially oriented site near the chromophore-binding site at 100K,
as observed by low-temperature X-ray crystallography and cryo-infrared crystal-
lography. Structural intermediates in the subsequent relaxation pathway involve
motion of CO 2 , amino acids and H-bonded waters both in the chromophore vicinity
and at longer range. This review provides an overview of the molecular character-
isation using structural and spectroscopy methods of this photoconversion reaction
of GFP. In addition, the mechanisms of photochromic reactions of mutants of GFP
and related fluorescent proteins will be summarised and discussed. These include
the cis - trans isomerisation and protonation changes in Dronpa, asFP595 and IrisFP
and related proteins, light-induced maturation in aceGFPL, and photoinduced beta-
elimination and backbone cleavage that leads to “green-to-red” photoconversion in
EosFP, Kaede, IrisFP and KikGR.
J.J. van Thor
Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London
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