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clearly points towards an at least two-step mechanismwith a nonradiative - possibly
very effectively quenched - intermediate.
Besides the I-form, which is blue shifted relative to the dominant B-form, a
number of emission spectra (6% of all sampled single molecules) were found for
EYFP that showed emission peaks red shifted well beyond 540 nm. The wide
spread of the maximum positions and the comparatively low occurrence made the
assignment of different forms difficult since no clear distribution of maximum
positions could be identified. Nevertheless, it was postulated that the observed
emission spectra belonged to three, so far unknown, spectral forms (Fig. 6 ). The
recorded spectral series showed transitions from the dominating B-form to the red-
shifted forms, evidencing the connection between these forms. Upon closer
inspection, the recorded emission spectra from these red-shifted forms showed
striking similarities to emission spectra recorded from DsRed group of proteins
(for details see below). Further, comparable red-shifted forms occurring with
comparable frequencies were found for EGFP, indicating a generic origin of all
these forms.
Besides the VFPs that belong to the GFP group of proteins, the proteins of the
DsRed group of proteins were intensely researched on the single molecule level
using spectrally resolved spectroscopy to identify different forms. The photophy-
sical complexity and multimeric nature is shared by many of the known red-
emitting proteins to date [ 10 , 16 , 82 - 84 ], which makes DsRed and its variants a
valuable model system for this group of proteins.
DsRed forms obligate tetramers [ 85 ] even at concentrations used for single
molecule spectroscopy, and the constituent monomeric subunits contain either a
green-emitting chromophore analogous to that of GFP or a red-emitting chromo-
phore [ 86 ]. Within the tetramers, the different chromophores are likely to be
coupled by fluorescence resonance energy transfer. This energy transfer coupling,
together with the intrinsic complexity seen for all VFPs, results in markedly rich
The first single molecule emission spectra from DsRed were presented as
evidence that immobilization of the proteins in a film of PVA does not change
the characteristic emission [ 74 ]. Already the first study of a statistically relevant
number of DsRed single molecule emission spectra highlighted the photophysical
complexity of DsRed [ 77 ]. The histogram of the single molecule emission maxi-
mum positions yielded the expected Gaussian distribution originating from spectral
diffusion. However, the distribution was clearly red shifted with respect to the bulk
emission maximum position, providing evidence for the fast formation of a red-
shifted, so-called super-red form under typical single molecule detection condi-
tions. The super-red form of DsRed was found to be photoinduced [ 47 , 87 ] and
single molecule experiments yielded emission spectra of this form for the first time
(Fig. 8 ).
These first pioneering studies were followed by systematic studies of the emis-
sion spectra of different DsRed variants at the single tetramer level that further
highlighted the spectral complexity of this group of proteins [ 88 ]. DsRed variants
with different amino acid substitutions (DsRed2: Arg2Ala, Lys5Glu, Lys9Thr,
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