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and ECFP (contains W57 and, in addition, W66 as part of the chromophore) two and
four resonances appear, respectively. Thus, the fluorinated Trp residues exist in two
states characterized by different broadening of the peaks, which indicates slow
exchange processes between the two states. Although Seifert et al. did not provide
detailed description for the Trp57 substitutes, the set of double signals cannot be
explained by the phenomena of dimerization or aggregation. This was demonstrated
by measurements with different labeled protein concentrations that resulted in
identical spectra. In addition, the 19 F NMR spectrum of ECFP[(6-F)Trp] in the
denatured state exhibits two resonances for (6-F)Trp57 as well. Most probably,
proline cis / trans conformations in the P56-W57-P58 sequence are the main cause
for the observed spectral behaviors. Interestingly, incorporation of (4-F)Trp and
(6-F)Trp into GFPuv did not result in double peaks in 19 F NMR and led only to
marginal differences in spectral shapes.
Most recently, it was shown that expression of ECFP in the presence of (4-Aza)
Trp and (7-Aza)Trp (see Fig. 1 ) yielded in high amounts of insoluble nonfluorescent
protein [ 59 ]. The small fraction of soluble protein has similar properties as the
parent protein but the fluorescence signal is much broader. Control experiments
were performed with EGFP, which confirmed that position 57 in the GFP molecule
is a crucial determinant of the folding process. Both ECFP and EGFP containing
either (4-Aza)Trp or (7-Aza)Trp were characterized by significantly higher
amounts of insoluble protein compared to the parent proteins (up to 90% insoluble
fraction). All attempts to perform proper refolding of these insoluble fractions
failed, although their solubilization was possible. It is worth to note that proteins
refolded in this way did not exhibit any defined secondary structure as revealed by
NMR. In addition, ESI-MS as well as spectroscopic analyses of these samples
indicated a complete Trp substitution by its aza-analogs but chromophore formation
did not take place. However, soluble chromophore-free av GFP mutants are already
reported. For instance, cis / trans isomerization of Xxx-Pro bonds can be affected by
mutagenesis of Xxx [ 60 ], which leads to hindered chromophore formation.
Interestingly, in case of the (Aza)Trp variants of ECFP and EGFP, the small
soluble protein fractions turned out to be mixtures of parent protein and variant.
High-performance liquid chromatography (HPLC) separation of these mixtures
revealed remarkable differences in retention times. Thus, the hydropathy of the
whole protein is changed by introduction of either only one (EGFP) or two (ECFP)
hydrophilic azatryptophans, although the fluorescence profiles of these species
were not affected significantly. Again, this strongly indicates that the correct
establishment of the protein's tertiary structure is much more important than the
composition of the tripeptide sequence (Xxx65-Xxx66-Xxx67) for the chromo-
phore formation [ 61 , 62 ].
As ECFP and EGFP give similar amounts of insoluble protein upon azatrypto-
phan incorporation, position 57 must be the crucial residue as it is the single Trp/
(Aza)Trp position (aside from W66 in ECFP). Trp57 is part of the PVPWP motif
and therefore involved in the water shielding protection of the chromophore from
solvent water [ 45 ]. Corresponding to the literature, the Trp substitution may affect
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