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To estimate changes in the flexibility of the chromophore environment, the exact
nature of the mutations in the chromophore environment and a model that linked the
flexibility of the chromophore environment with the speed of chromophore forma-
tion was considered for each variant. The narrowest distribution of maximum
positions was found for the protein variant DsRed2, which had the largest chromo-
phore environment flexibility. For the other protein variants, the increase in distri-
bution width of maximum positions corresponded to decreasing chromophore
environment flexibility. Clearly, a more rigid chromophore environment results in
a broader distribution of the emission maximum positions, that is, exhibits increased
spectral diffusion. It was postulated that the correlation between the width of the
distribution of single molecule emission maximum positions and the flexibility of
the chromophore environment reflects the ability of the protein to return to the
energetically most favorable conformation after, for example, a thermally induced
reorientation. Flexible systems can rapidly reach the global energy minimum while
in more rigid systems the return to the global energy minimum goes via local
minima that act as traps and that would have different emission spectra.
Although the chemical environment of the chromophore showed clear differ-
ences from variant to variant, these differences in the chemical environment did not
break the correlation between distribution width and rigidity of the chromophore
environment. It was concluded that in this fluorescent protein system these chemi-
cal variations close to the chromophore only played a minor role for the observed
spectral diffusion.
Identification and Characterization of Distinct Spectral Forms
Chemical dyes are purified before use to ensure an ensemble of chemically identical
emitters. The purification of VFPs in a similar fashion is not possible. Due to the
complex autocatalytic processes forming the fluorescent chromophore potentially
resulting in different chromophores, the influences of chromophore maturation, and
the varying effects of the protein that is encapsulating the chromophore, VFP
ensembles are essentially complex mixtures of photophysically different suben-
sembles. Additionally, these forms might be in equilibrium with each other and
exhibit interchanges between the different forms.
To discriminate between spectral diffusion and different spectral forms, the most
straightforward approach is to analyze a statistically relevant number of molecules
and to subsequently create histograms of the relevant observable. In the histogram
spectral diffusion results in a unimodal distribution. Different spectral forms are
apparent as distinct peaks in a multimodal distribution. Each spectral form is
defined by a characteristic central value, which is the most likely value of the
relevant observable, and a distribution width, which is defined by spectral diffusion.
Since each spectral form is subject to spectral diffusion, large spectral distributions
generally point toward the presence of different spectral forms, while variations
originating solely from spectral diffusion are usually small [ 69 ].
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