particular tertiary structure plays a crucial role both in formation/maturation of the
chromophore and in regulating its photophysical properties.
2.2 The Chromophore
The chromophore forms autocatalytically from a tripeptide (in GFP: Ser65-Tyr66-
Gly67), requiring nothing else but molecular oxygen. In general, the first amino
acid may be any one, but the second and third amino acids, tyrosine and glycine, are
strictly conserved in nature. However, while glycine is absolutely crucial for
chromophore self-synthesis, tyrosine can be replaced, e.g., by histidine, phenylala-
nine, or tryptophan [ 5 ].
The green GFP chromophore, 4-( p -hydroxybenzylidene)-5-imidazolinone
( p -HBI), results from a sequential cyclization-oxidation-dehydration reaction, with
characteristic time scales ranging fromminutes to hours (Fig. 1b ). In a first step during
protein folding, a peptide cyclization and possibly proton transfers occur, and a
heterocyclic intermediate forms within a few minutes. In a second step, the protein
reacts with O 2 to oxidize the tyrosine C
bond, and to generate a cyclic imine,
hydrogen peroxide is released. The final step again involves a proton transfer reaction
that induces bond rearrangements and leads to the fully conjugated, mature chromo-
phore [ 17 ]. In the planar p -HBI chromophore, the conjugated
extends from the p -hydroxybenzyl ring of the tyrosine to the imidazolinone ring.
With respect to the double bond of the methene bridge (C
of Tyr66), both rings
are arranged in a cis conformation. Real-time visualization of de novo synthesis and
folding of GFPmolecules using single-molecule fluorescence microscopy has demon-
strated that the fluorescence of the fastest GFP molecules appeared already within
1min[ 18 ]. Because of the technique used, GFP molecules become detectable only
after chromophore formation. Therefore, the characteristic time obtained from these
experiments is related to the overall sequence of polypeptide synthesis, protein
folding, and chromophore formation. In bulk measurements, transition midpoints of
~30 to ~90 min were observed for the maturation of GFP [ 5 ].
The absorption spectrum of wild-type GFP displays two bands associated with
the chromophore (Fig. 1c ). The dominant A band at 395 nm and the weaker B band
at 475 nm represent the neutral and anionic tyrosine moieties, respectively. Upon
excitation of the B band, the fluorescence emission peaks at 508 nm in the green
(Fig. 1c ). Interestingly, excitation of the neutral chromophore also results in green
emission of an anionic species: in the excited state, the phenolic proton is released
and transferred to Glu222 (excited state proton transfer, ESPT), so that the anionic
species forms before photon emission [ 19 - 21 ].
2.3 Variations of the Chromophore Motif
In recent years, a number of modifications of the p -HBI chromophore have been
found in GFP-like proteins from natural sources. In orange and red-fluorescent