Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa

Satoshi Habuchi, Ryoko Ando, Peter Dedecker, Wendy Verheijen, Hideaki Mizuno, Atsushi Miyawaki, Johan Hofkens*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

364 Scopus citations

Abstract

Reversible photoswitching of individual molecules has been demonstrated for a number of mutants of the green fluorescent protein (GFP). To date, however, a limited number of switching events with slow response to light have been achieved at the single-molecule level. Here, we report reversible photoswitching characteristics observed in individual molecules of Dronpa, a mutant of a GFP-like fluorescent protein that was cloned from a coral Pectiniidae. Ensemble spectroscopy shows that intense irradiation at 488 nm changes Dronpa to a dim protonated form, but even weak irradiation at 405 nm restores it to the bright deprotonated form. Although Dronpa exists in an acid-base equilibrium, only the photoinduced protonated form shows the switching behavior. At the single-molecule level, 488- and 405-nm lights can be used to drive the molecule back and forth between the bright and dim states. Such reversible photoswitching could be repeated >100 times. The response speed to irradiation depends almost linearly on the irradiation power, with the response time being in the order of milliseconds. The perfect reversibility of the Dronpa photoswitching allows us to propose a detailed model, which quantitatively describes interconversion among the various states. The fast response of Dronpa to light holds great promise for following fast diffusion or transport of signaling molecules in live cells.

Original languageEnglish (US)
Pages (from-to)9511-9516
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number27
DOIs
StatePublished - Jul 5 2005

Keywords

  • Fluorescence microscopy
  • Photochromism
  • Protonation/deprotonation

ASJC Scopus subject areas

  • General

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