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

Mircea Cotlet, Johan Hofkens*, Satoshi Habuchi, Gunter Dirix, Maarten Van Guyse, Jan Michiels, Jos Vanderleyden, Frans C. De Schryver

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

135 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)14398-14403
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number25
DOIs
StatePublished - Dec 4 2001

ASJC Scopus subject areas

  • General

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