Spectral Versatility of Fluorescent Proteins Observed on the Single Molecule Level - Fluorescent Proteins: From Understanding to Design - page 225

Fig. 8 Emission spectra from
single DsRed tetramers.
Emission with maximum at
583 nm as observed for
ensemble samples was only
rarely observed. Most of the
emission spectra showed a
distinct shift of the emission
spectra to longer
wavelengths. This red-shifted
emission was attributed to the
photoinduced formation of a
“super-red form” (From [ 77 ],
Copyright (2001) National
Academy of Sciences, USA)
Val105Ala, Ile161Thr, Ser197Ala; Fluorescent Timer: Val105Ala, Ser197Thr;
DsRed_N42H: Asn42His; AG4: Val71Met, Val105Ala, Ser197Thr) exhibiting
altered spectral and maturation properties were thoroughly analyzed. In these
studies, single molecule emission spectra from the green-emitting DsRed chromo-
phore were measured for the first time. Full sets of emission spectra consisting of
spectra from the green-emitting chromophore and from the red-emitting chromo-
phore were presented for DsRed as well as for all sampled variants (Fig. 9 ). Also
single protein tetramers were found that showed mixed emission from the green as
well as from the red-emitting chromophore, providing direct evidence of the
existence of mixed tetramers consisting of tetramers containing the different chro-
mophores.
To identify different spectral forms and especially to establish whether the
super-red form observed for DsRed can be found for all DsRed variants, the
emission maximum positions were determined and assembled into histograms.
The analysis of the emission maximum positions of all single tetramer spectra of
all variants showed that the emission maximum positions of the green-emitting
chromophores are distributed around ~503 nm, consistent with bulk spectroscopy
(Fig. 9 ). The emission maximum positions of the red chromophores were found to
be distributed ~600 nm (with slight differences for the different variants), a value
that is ~16 nm red shifted relative to the bulk emission maxima. This observation
verified the rapid formation of a high quantum efficiency super-red form for all
DsRed variants.
Although the super-red form has been observed for DsRed and a number of
variants, its underlying molecular origins are still unclear. It has been suggested
that the photoconversion to the super-red form originates from structural changes
in the vicinity of the chromophore due to a cis-trans isomerization of the
chromophore and a decarboxylation of a glutamate (Glu-215) in the chromophore
vicinity [ 46 ]. These changes result in a red-shifted, low quantum efficiency form.