Biology Reference
In-Depth Information
Fig. 2 Photophysical properties and structure of GdFP in comparison with common av GFP
mutants. Normalized absorbance (a) and fluorescence (b) spectra of GdFP, ECFP, EGFP and
EYFP. While ECFP (Trp66) has two characteristic absorbance maxima at 434 and 452 nm, GdFP
((4-Am)Trp66) has only one, but red-shifted maximum at 466 nm. The absorbance spectra of EGFP
and EYFP are further red-shifted to 488 and 512 nm, respectively. In contrast, GdFP shows the
most red-shifted fluorescence (l max
574 nm) when compared to all av GFP variants produced by
traditional DNA technologies. ECFP, EGFP and EYFP only have maximum fluorescence wave-
length of 476 (505), 509 and 527 nm, respectively. Detailed information about maximum absor-
bance and fluorescence wavelengths of the different FPs can be found in Table 1 and in the text.
(c) Structural overlay of GdFP ( yellow ) with EGFP ( green ) and ECFP ( blue ). The chromophore and
residues Tyr145 and His148 are represented as sticks in the same colors with the exception of
ECFP, where blue (conformation A 0 of ECFP) and red (conformation B 0 of ECFP) are used
The same effect is observed if the chromophore is additionally increased by
conjugation with a fourth amino acid as present for example in ds Red [ 18 ]. GdFP,
however, maintains the basic p -HBI structure of the av GFP chromophores but also
exhibits an emission wavelength comparable to the more extended ds Red chromo-
phore. One possible explanation for such a significant change in spectral properties
was the emergence of intramolecular charge transfer processes (ICTs): the intro-
duced amino group is acting as an additional electron donor and is conjugated to the
-system of the tryptophan moiety. This leads to a resonance stabiliza-
tion of the excited state explaining the vast red-shift in the emission spectrum [ 11 ].
2.2.2 Stokes Shift
The chromophore of GdFP is not only characterized by a large red-shift in absor-
bance and fluorescence spectra when compared to ECFP but also by a Stokes shift
Search WWH ::

Custom Search