Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: Mechanism responsible for the reversible photoswitching

Satoshi Habuchi, Peter Dedecker, Jun ichi Hotta, Cristina Flors, Ryoko Ando, Hideaki Mizuno, Atsushi Miyawaki, Johan Hofkens*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

81 Scopus citations


Recently, reversible photoswitching in bulk samples or in individual molecules of Dronpa, a mutant of a green fluorescent protein (GFP)-like fluorescent protein, has been demonstrated. Intense irradiation at 488 nm changed Dronpa in a dim protonated form, and weak irradiation at 405 nm restored it to the bright deprotonated form. Here, we report on the mechanism of photoswitching of Dronpa by means of ensemble and single-molecule spectroscopy. Ensemble spectroscopy shows that the photoswitching can be described, in first approximation, by a three-state model including a deprotonated (B), a protonated (A1), and a photoswitched protonated (A2) forms of the chromophore. While the B and the A1 forms are in a ground state acid–base equilibrium, the B and the A2 forms are reversibly photoswitched upon irradiation with 488 and 405 nm light. At the single-molecule level, the on-times in fluorescence intensity trajectories excited at 488 nm decrease with increasing the excitation power, consistent with the photoswitching from the B to A2 form. The on-times agree well with expected values, which are calculated based on the ensemble spectroscopic properties of Dronpa. The fluorescence trajectory obtained with simultaneous dual-color excitation at 488 and 405 nm demonstrates reversible photoswitching between the B and the A2 forms at the single-molecule level. The efficiency of the photoswitching from the A2 to B form increased with increasing the excitation power of the 405 nm light. Our results demonstrate that Dronpa holds its outstanding photoswitching properties, based on a photo-induced protonation/deprotonation process, even at the single-molecule level.

Original languageEnglish (US)
Pages (from-to)567-576
Number of pages10
JournalPhotochemical and Photobiological Sciences
Issue number6
StatePublished - Jun 7 2006
Externally publishedYes

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry


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