Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy

The photophysics and photochemistry taking place in the DsRed protein, a re cently cloned red fluorescent protein from a coral of the Discosoma genus, are investigated here by means of ensemble and single-molecule time-resolve d detection and spectroscopic measurements. Ensemble time-resolved data rev eal that 25% of the immature green chromophores are present in tetramers co ntaining only this immature form. They are responsible for the weak fluores cence emitted at 500 nm. The remaining 75% of the immature green chromophor es are involved in a fluorescence resonance energy transfer process to the red species. The combination of time-resolved detection with spectroscopy a t the single-molecule level reveals, on 543-nm excitation of individual DsR ed tetramers, the existence of a photoconversion of the red chromophore emi tting at 583 nm and decaying with a 3.2-ns time constant into a super red o ne 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 leve l by the observation of the creation of a super red form and a blue absorbi ng species on irradiation with 532-nm pulsed light at high excitation power . Furthermore, single-molecule experiments suggest that a similar photoconv ersion process might occur in the immature green species on 488-nm excitati on.