Reversible Single-Molecule Photoswitching in the GFP-Like Fluorescent Protein Dronpa

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 photo...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 27; pp. 9511 - 9516
Main Authors Habuchi, Satoshi, Ando, Ryoko, Dedecker, Peter, Verheijen, Wendy, Mizuno, Hideaki, Miyawaki, Atsushi, Hofkens, John, Hastings, J. Woodland
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 05.07.2005
National Acad Sciences
SeriesFrom the Cover
Subjects
Absorption spectra
Animals
Anthozoa - chemistry
Biological Sciences
Evolution
Fluorescence
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - radiation effects
Irradiation
Laser power
Lasers
Light
Line spectra
Models, Chemical
Molecular excitation
Molecules
Pectiniidae
Photochemistry
Proteins
Spectrometry, Fluorescence - methods
Time Factors
Trajectories
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Summary: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.
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Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, SGM209, Boston, MA 02115.
See Commentary on page 9433.
Abbreviations: ESPT, excited-state proton transfer; PVA, poly(vinyl alcohol).
To whom correspondence should be sent at the § address. E-mail: hofkens@chim.ucl.ac.be or johan.hofkens@chem.kuleuven.be.
This paper was submitted directly (Track II) to the PNAS office.
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.
Edited by J. Woodland Hastings, Harvard University, Cambridge, MA
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0500489102