which optimize the Trp chromophore accommodation within the GFP scaffold,
good fluorescence intensities were achieved [ 7 ].
In general, av GFP derivates are monomers when expressed heterologously since
the wild-type av GFP has an only weak tendency to dimerize. In contrast, most other
FPs are naturally occurring as multimers, which strongly limit their applications in
cell and molecular biology. To make these proteins available for general use, a great
amount of engineering had to be performed. For example, the first isolated red FP
ds Red [ 10 ] had to be mutated at not less than 45 positions to obtain the now widely
used ds Red-monomer (Clontech).
The need of vast protein optimization in case of many FPs demonstrates the need
to explore other strategies for the development of novel spectral classes of FPs as
well. One strategy is the use of noncanonical amino acids (NCAAs) because
classical protein engineering methods are restrained to the standard genetic code.
Aromatic noncanonical residues are especially interesting for FP engineering
because a higher amount of variation can be introduced at position 66 (with the
standard genetic code only His, Tyr, Phe, and Trp are available). A greater variety
of substituted aromatic residues with interesting spectral and other biophysical
properties are nowadays available for FP in vivo expression, mainly in bacterial
cells (see Fig. 1 ). This chapter will try to give an overview of the works exploring
the effects of NCAAs on FP structure and biophysical properties. All abbreviations
and structures of the used NCAAs are summarized in Fig. 1 .
2 Chromophore Redesign
2.1 Chromophore with 4-Aminotryptophan
Among all noncanonical indole moieties tested for Trp66 substitution in ECFP to
date, the greatest change in spectral properties was delivered by the indole
4-aminoindole substitution (see Fig. 1 ) in the chromophore of ECFP [ 11 ]. The
resulting protein was named gold fluorescent protein (GdFP) and exhibited maximal
absorbance and emission at 466 and 574 nm, respectively. This makes GdFP the most
red-shifted av GFP variant with the largest Stokes shift (108 nm) known to date. Its
fluorescence is 69 nm red-shifted when compared to the parent ECFP and 47 nm
when compared to the emission maximum of EYFP (see Fig. 2 ). In addition, GdFP is
also characterized by increased thermostability and decreased aggregation tendency.
2.2 Spectroscopic Features of GdFP
2.2.1 Red-Shift in Absorbance and Fluorescence
From the different classes of neutral av GFP chromophores, it is known that an
increase in size of the delocalized
-system results in a corresponding increase in
excitation and emission wavelengths (see Table 1 ).