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since the polymer matrix embedding the chromophore is not exactly defined. In
contrast to chromophores embedded in a polymer, the local environment of the
chromophore of a VFP is precisely defined by the amino-acid composition of the
protein scaffold encapsulating the chromophore. However, this strict definition of
the chromophore environment is not sufficient to ensure invariant spectral charac-
teristics of the VFPs. Indeed at the single molecule level, VFP photophysical
parameters have been found to be distributed as in any other system of chromo-
phores embedded in a matrix. Thus, even in these protein systems that possess a
chemically well-defined nanoenvironment around the chromophore, the observed
spectral heterogeneity reflects the subtle structural variations within the protein that
influence the chromophore nanoenvironment and lead to distinct chromophore-
matrix interactions.
By site-directed mutagenesis in and around the chromophore the chemical
environment of the chromophore in a VFP can be precisely altered. Genetic
engineering has yielded a wide range of mutant VFPs, such that a whole palette
of proteins with different chromophore environments is available. Based on these
variants it was possible to analyze the effect on spectral diffusion of interactions
between the chromophore and its environment. Since some VFPs are capable of
forming chemically different chromophores emitting at different wavelengths
within one and the same scaffold, one can not only analyze changes in the environ-
ment of the chromophore defined by the protein, but also characterize spectral
diffusion of different chromophores within identical nanoenvironments.
Spectral diffusion of VFPs was researched in detail in a comparative study on the
tetrameric reef coral fluorescent protein DsRed and a number of its variants. As
the first discovered red-emitting VFP, DsRed has been widely studied [ 17 , 19 , 20 ,
40 , 75 - 79 ]. DsRed was extensively engineered to yield variants with changed and
optimized properties. In DsRed, the autocatalytic reaction that forms the chromo-
phore within the protein can branch and result in two different end products,
yielding either a green-emitting or red-emitting chromophore. Different variants
of DsRed exhibiting different green to red chromophore ratios have been generated,
yielding proteins that are essentially all green emitting, all red emitting, or exhibit-
ing mixed green and red emission. For this study, a large number of single molecule
emission spectra were collected from DsRed, DsRed2, and Fluorescent timer (all
predominantly red emitting), DsRed_N42H (mixed green and red emission), and
AG4 (mainly green emitting) (see Fig. 4 )[ 51 ].
Spectral diffusion was characterized by measuring the emission maximum posi-
tion. The exact position of the emission spectrum is easy to determine and the
emission maximum position has been found to be especially sensitive to changes in
the local chromophore environment [ 42 , 49 - 52 ]. To precisely determine the emis-
sion maximum position, a double Gaussian was fitted to each emission band in the
single molecule spectra. From the individual emission maximum positions, histo-
grams were constructed that showed the distribution of emission maximum posi-
tions for each protein variant (Fig. 5 ).
The width of the distribution was found to be characteristic for each chromo-
phore-matrix combination and independent of experimental parameters such as the
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