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[ 11 ]. However, the above described tremendous red-shift and extreme large Stokes
shift of the ECFP[(4-Am)Trp] variant (called golden fluorescent protein GdFP) is
only due to (4-Am)Trp at position 66, whereas expectedly (4-Am)Trp57 does not
affect the spectral properties of the protein.
Furthermore, the chromophore seems to be tightly packed in GdFP and the
crystalline state possesses a rather rigid architecture as there is no additional
electron density indicating different conformational states. In addition, the region
of residues Tyr145-Asn149 shows no flexibility as it is, for instance, the case in the
“minor” conformation of ECPF. In contrast, whereas the ECFP chromophore
exhibits eight mainly hydrophobic interactions with residues in its vicinity and
one hydrogen bond (to Ser205), the chromophore of GdFP interacts only with five
neighboring residues [ 11 ]. However, the amino group of (4-Am)Trp66 may interact
with Phe165. This would enlarge the interacting network of aromatic residues
Phe165-His148-Tyr145. These residues are rather rigid and involved in hydropho-
bic contacts with the chromophore. This enhanced network interaction would also
explain the higher stability of GdFP compared to ECFP and its lower tendency for
aggregation as well as its more co-operative unfolding process.
As already mentioned, azatryptophan inhibits proper folding of EGFP/ECFP and
chromophore formation ( vide supra )[ 59 ]. In contrast, chalcogen-containing Trp
analogs ([2,3]Sep, [3,2]Sep, [2,3]Tpa and [3,2]Tpa) support proper folding of ECFP
as the protein is mainly expressed in the soluble fraction and shows the same
electrophoretic mobility as the parent protein in native two-dimensional protein
gel analysis. But, on the contrary, the variants do not exhibit the characteristic
fluorescence properties even if they are colored at daylight [ 15 ]. The fact that these
ECFP variants are colored suggests chromophore formation but quenched fluores-
cence. Unfortunately, mass spectrometry only revealed a high heterogeneity of the
Expectedly, these analogs cause no shifts of the fluorescence emission maximum
in EGFP with single replacement at position 57. The proteins are expressed in high
yields, which enabled their crystallization and X-ray structure elucidation. Expect-
edly, crystal structures of EGFP[3,2]Tpa, EGFP[2,3]Sep and EGFP[3,2]Sep
revealed the same overall topology as EGFP, and no significant changes in the
environment of residue 57 (Fig. 12 )[ 15 ]. However, incorporation of [3,2]Tpa into
EGFP increased the absorption and fluorescence emission intensity by approx.
25%. This is comparable with the effects of in vitro incorporation of selenomethio-
nine into av GFP or the introduction of Met into position 69 into EYFP [ 32 ]. With
chalcogen-containing Trp analogs, this effect occurs solely in case of [3,2]Tpa but
not [2,3]Tpa or in the Sep-variants of EGFP. Therefore, this must be an electrostatic
effect in combination with a particularly favorable geometric arrangement of the
chromophore's vicinity [ 15 ]. This is reasonable as the distance between the sulfur
of Tpa57 and sulfur of M218 is 4.7 ˚ for [3,2]Tpa but 6.6 ˚ for [2,3]Tpa. In
addition, even if the sulfur-selenium distance for M218 and [3,2]Sep57 is also
4.7 ˚ , [3,2]Sep does not cause the same effect as [3,2]Tpa.
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