Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa
- Satoshi Habuchi*,†,
- Ryoko Ando‡,
- Peter Dedecker*,
- Wendy Verheijen§,
- Hideaki Mizuno‡,
- Atsushi Miyawaki‡, and
- Johan Hofkens*,§,¶
- *Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; ‡Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; and §Unité Chimie des Matériaux Inorganiques et Organiques, Université Catholique de Louvain, Bâtiment Lavoisier Place L. Pasteur 1, 1348 Louvain-la-Neuve, Belgium
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Edited by J. Woodland Hastings, Harvard University, Cambridge, MA (received for review January 19, 2005)
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.
Footnotes
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↵ ¶ To whom correspondence should be sent at the § address. E-mail: hofkens{at}chim.ucl.ac.be or johan.hofkens{at}chem.kuleuven.be.
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↵ † Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, SGM209, Boston, MA 02115.
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Author contributions: S.H., A.M., and J.H. designed research; S.H. performed research; R.A., H.M., and A.M. contributed new reagents/analytic tools; S.H., W.V., and P.D. analyzed data; S.H., A.M., and J.H. wrote the paper; and H.M. provided helpful discussion.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: ESPT, excited-state proton transfer; PVA, poly(vinyl alcohol).
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See Commentary on page 9433.
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↵ ∥ The photoswitched protonated form and the protonated form that is in equilibrium with the deprotonated form are the different species, as discussed later. A detailed analysis of the time evolution of the absorption spectrum that takes into account the acid–base equilibrium is given in Supporting Text.
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↵ ** Here, ESPT is defined as proton transfer between the protonated and the deprotonated form in the excited state. The photoswitching from the protonated and the deprotonated form would, therefore, take place from the excited state of the protonated form to the ground state of the deprotonated form. More likely, the process goes over an intermediate in the excited state and from the intermediate form to the ground state.
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↵ †† Recovery of the fluorescence on 405-nm irradiation is not perfect in Fig. 2g (≈90%). This result is probably due to insufficient irradiation of 405-nm light to recover the deprotonated state or insufficient time that the molecule spent in the deprotonated state to get detectable photon numbers.
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Freely available online through the PNAS open access option.
- Copyright © 2005, The National Academy of Sciences
