Abstract
The photophysics and photochemistry taking place in
the DsRed protein, a recently cloned red fluorescent protein from a
coral of the
Discosoma
genus, are investigated here by
means of ensemble and single-molecule time-resolved detection and
spectroscopic measurements. Ensemble time-resolved data reveal that
25% of the immature green chromophores are present in tetramers
containing only this immature form. They are responsible for the weak
fluorescence emitted at 500 nm. The remaining 75% of the immature
green chromophores are involved in a fluorescence resonance energy
transfer process to the red species. The combination of time-resolved
detection with spectroscopy at the single-molecule level reveals, on
543-nm excitation of individual DsRed tetramers, the existence of a
photoconversion of the red chromophore emitting at 583 nm and decaying
with a 3.2-ns time constant into a super red one emitting at 595 nm and
for which the decay time constant ranges between 2.7 and 1.5 ns. The
phenomenon is further corroborated at the ensemble level by the
observation of the creation of a super red form and a blue absorbing
species on irradiation with 532-nm pulsed light at high excitation
power. Furthermore, single-molecule experiments suggest that a similar
photoconversion process might occur in the immature green species on
488-nm excitation.