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selenomethionine in the vicinity of the chromophore, similar findings were reported
[ 31 , 32 ].
With BT (see Fig. 1 ), another Sulfur-containing Trp analog was recently intro-
duced site specifically into CFP6 (see Sect. 2.3.1 ) by Tirrell and co-workers. CFP6
[BT] showed blue shifts of 34 and 16 nm in the absorption and fluorescence
emission maxima, respectively. This is congruent with the data obtained from
incorporation of [2,3]Tpa and [3,2]Tpa. Furthermore, the chromophore's extinction
coefficient was similarly lowered by BT incorporation (BT: 8,100 M 1 cm 1 , [2,3]
Tpa: 6,840 M 1 cm 1 and [3,2]Tpa: 6,350 M 1 cm 1 ). However, in contrast to
[2,3]Tpa- and [3,2]Tpa-containing ECFP, CFP6[BT] exhibited fluorescence emis-
sion and showed a relatively high Stokes shift of 56 nm (CFP6: 37 nm) with only
threefold reduction in quantum yield when compared to CFP6.
Tirrell and co-workers argued that CFP6[BT] could be used as a FRET partner
for GFP since its fluorescence spectrum overlaps with the GFP's excitation spec-
trum and it can be efficiently excited with a violet diode laser.
3 NCAA Incorporation and Structural Integrity of FPs
Introduction and General Remarks
Besides the spectroscopic properties, structural integrity of a fluorescent protein
may change upon incorporation of NCAAs. On the contrary, structural details of
this protein family can be visualized and investigated from a different perspective
with the help of NCAAs. Therefore, in the following the effects of NCAA incor-
poration into av GFP and its mutants will be discussed in the light of information
gained mainly from high-resolution X-ray structures.
The canonical tertiary structure of av GFP and all its mutants is built from 11
-strands and a central helix holding the chromophore in the context of a central
cavity, which is solvent inaccessible. This basic structure, the so-called
fold, is maintained in all soluble mutants. Another feature of the GFP family is the
chromophore, which is generated through a self-catalyzed intramolecular posttrans-
lational modification (see Introduction). However, chromophore formation is not an
intrinsic property of the tripeptide but rather of the proper folding of the surround-
ing protein matrix. This is further evidenced by several observations, e.g., dena-
tured GFP or chemically synthesized model chromophores do not show the
characteristic fluorescence at ambient temperatures. In contrast, high fluorescence
of the isolated chromophore is detectable at 77 K [ 33 , 34 ]. Thus, chromophore
fluorescence at ambient temperature is possible only in the context of its solvent-
free and rigid protein cavity, and its general optical properties are very sensitive to
mutations of surrounding residues [ 35 , 36 ]. From this point of view, it is easy to
anticipate the GFP family as a potent model for NCAA incorporation experiments.
Surprisingly, all known fluorescent proteins with an av GFP-like
-barrel fold exhibit
only four conserved residues. In the av GFP primary sequence, these residues are Tyr66,
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