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Fig. 2 The GFP chromophore and its autocatalytically produced variants (adopted from [ 24 ])
proteins such as DsRed [ 22 ] and eqFP611 [ 23 ], the single bond between the amide
nitrogen and the C
atom of the first of the three chromophore-forming amino acids
is oxidized to an acylimine group in-plane with the p-HBI chromophore (Fig. 2 ).
Consequently, the conjugated
-electron system of the resulting 2-imino-5-(4-
hydroxybenzylidene)-imidazolinone chromophore is further extended, and the
emission shifts bathochromically to ~600 nm. The delocalized
-electron system
may extend even further to include the carbonyl group of the preceding amino acid
in FPs emitting further to the red [ 25 ].
In mOrange, a monomeric DsRed variant emitting at 562 nm, an oxazole
heterocycle (Fig. 2 ) is created from the side chain of Thr66, the first amino acid
of the chromophore triad in this protein [ 26 ]. Its hydroxyl group apparently attacks
the preceding carbonyl carbon. The yellow zFP538 from Zoanthus sp. likewise
features a third heterocycle, formed by ring closure of a lysine side chain [ 27 ].
Presumably, the terminal amino group of the lysine side chain attacks the reactive
acylimine bond and, concomitantly, the protein backbone is cleaved, leav-
ing the N-terminal fragment with a terminal carboxamide (Fig. 2 ). The smaller red
shift compared with the acylimine of DsRed has been rationalized by the less
effective charge delocalization by the cyclic imine [ 27 ]. In AsRed, the mutant
Ala143Ser of asFP595 from Anemonia sulcata [ 28 ], the X-ray structure analysis
revealed that chromophore maturation is accompanied by a break in the polypeptide
chain [ 29 - 31 ], and a carbonyl group, generated by hydrolysis of the intermediately
formed acylimine group, extends the conjugated
-electron system of the p -HBI
chromophore (Fig. 2 ).
In addition to these spontaneously occurring chemical alterations of the green
chromophore, photoinduced modifications can occur, as will be discussed in detail
in Sect. 3.5.2.
2.4 Quaternary Structure
GFP has only a weak tendency to form dimers; the dissociation coefficient has been
estimated as 100
M[ 32 ]. The homodimeric interface includes a small patch of
hydrophobic residues (Ala206, Leu221, Phe223) as well as a large number of
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