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The histogram of the emission maximum positions from single EYFP molecules
was clearly not a unimodal Gaussian distribution. The distribution is very widely
stretched with a dominating peak around ~527 nm. This peak was expected, since
the predominant form in EYFP was known from ensemble studies to be the B-form
with emission maximum at 527 nm. More interesting were the secondary distribu-
tions on the blue as well as on the red side of the main distribution that suggested the
existence of different, coexisting spectral forms. On the blue side, a secondary
distribution centered at ~508 nm was found that was identified to originate from the
blue-shifted intermediate I-form that had heretofore only been observed at cryo-
genic temperatures. Using single molecule spectroscopy does not only provide
evidence of the presence of spectrally different forms but also allows the character-
ization of these forms without being disturbed by the overwhelming contribution of
the dominating forms as in ensemble studies. In case of EYFP, the single molecule
study resulted in the first undisturbed emission spectra of the I-form (Fig. 6 ), which
yielded interesting hints about the underlying molecular basis of this form. Inter-
estingly, the EYFP I-form spectrum was found to be congruent with the well-known
emission spectrum from the anionic GFP chromophore. The EYFP emission is red
shifted compared to the emission of the anionic GFP chromophore due to
stacking interactions between the aromatic ring of the tyrosine residue introduced
at position 203 and the chromophoric
-system [ 50 ]. Based on the observation that
the EYFP I-form spectrum resembles the emission of the chromophore lacking
stacking, it was concluded that a disturbed interaction of the
-stacking by a
conformational change in the chromophore surrounding, e.g., by a turn or tilt of
the phenolic group of the tyrosine residue most likely results in the spectral signature
observed for the I-form.
Since series of emission spectra from each molecule were sampled, it was also
possible to observe direct dynamics between the different forms - an observation
impossible in ensemble spectroscopy. Interestingly, the transition between the
B- and the I- form was always accompanied by a dark period between the disap-
pearance of one and the appearance of the other form (Fig. 7 ). This spectral signature
Fig. 7 Spectral sequence showing the transition from the predominant EYFP B-form to the blue-
shifted I-form. The transitions were always accompanied by a dark period between the observa-
tions of emission from the two different forms (Reprinted from [ 36 ], with permission from
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