Synthetic Biology of Autofluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 97

2.4 Chromophores with (4-Am)Phe, (O-Me)Tyr and (3-Am)Tyr . . . . . . . . . . . . . . . . . . . . . . . 110
2.5 Chromophores with Chalcogen Containing Trp Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3 NCAA Incorporation and Structural Integrity of FPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3.1 Introduction and General Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3.2 Aliphatic NCAAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
3.3 Aromatic NCAAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4 Applications and Further Development of Autofluorescent Proteins Using NCAAs . . . . . 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

1 Introduction

As explained in detail in the preceding chapters, a great variety of fluorescent
protein spectral classes was gained in the last years by protein engineering of
Aequorea victoria GFP ( av GFP, e.g., generation of ECFP) or “natural” isolation
from other species, mainly reef corals from the species Anthozoa, e.g., ds Red [ 1 ]. In
most naturally occurring fluorescent proteins, the chromophore is 4-( p -hydroxy-
benzylidene)-5-imidazolinone ( p -HBI) or a derivative with an extended -electron

system obtained by further autocatalytic reactions from a p -HBI intermediate [ 1 , 2 ].
While the p -HBI structure is obtained from a reaction between aa65, Tyr66, and
Gly67, the key feature of engineered av GFP variants is an exchange of the aromatic
Tyr residue by other canonical aromatic amino acids. The occurrence of an aro-
matic residue at position 66 is crucial for chromophore fluorescence. Thereby,
enhanced blue fluorescent protein (EBFP) was designed by introduction of His
and enhanced cyan fluorescent protein (ECFP) by introduction of Trp at position 66.
Y66F also gave rise to a functional chromophore; however, the fluorescence
characteristics were largely reduced in comparison to the other mutants and not
much effort was made to develop the mutant further. The mutants Tyr66

p

Phe/
His/Trp show a characteristic blue emission since the excited state proton transfer
(ESPT), responsible for the characteristic green fluorescence in native av GFP, is
prohibited [ 3 ]. The only av GFP derivative with a chromophore identical to av GFP
but significantly different spectral properties is enhanced yellow fluorescent protein
(EYFP). It was designed by the mutation T203Y leading to juxtaposition of Y203
and the phenolate anion of the p -HBI chromophore. This leads to

!

stacking
interactions stabilizing the excited state dipole moment and thus red-shifting the
excitation and emission maxima about 20 nm when compared to av GFP. According
to these findings, Roger Tsien subdivided the av GFP mutants into seven classes [ 4 ].
These were already discussed in great detail in the chapters by Nifosi and Tozzini,
Wiedenmann et al. and Jung et al.
It is often not considered that beyond the key mutations in the chromophore,
most av GFP derivates contain other crucial mutations responsible for lower aggre-
gation tendencies, better folding (e.g., GFPuv [ 5 ]; F99S, M153T, V163A) and
higher fluorescence intensities (e.g., EGFP [ 6 ], ECFP, EYFP [ 7 , 8 ]). The impor-
tance of such “secondary” mutations is best illustrated in case of ECFP. A sole
mutation of Y66W in av GFP indeed resulted in the characteristic absorbance and
fluorescence maxima of ECFP but the fluorescence intensity was negligible [ 9 ].
Only by introduction of three additional mutations (N146I, M153T and V163A),

p - p