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Dronpa-Green Fluorescent Protein

  • Photoswitchable fluorescent protein
  • Reversible “on-off” switching
  • Useful for measuring diffusion and transport of molecules in living cells

The fluorescent protein Dronpa gene was originally cloned from stony coral Echinophyllia sp. (Kikka-sango in Japanese).

CoralHue® Dronpa-Green1 (DG1) is a monomeric green fluorescent protein that is photochromic: its fluorescence is reversible, with “on-off” switching on demand through exposure to di fferent wavelengths of light. DG 1 bleaches immediately following strong excitation at -500 nm. Bleached DG1 completely regains its bright green fluorescence after irradiation at -400 nm. This unique property of DG1 is useful for repeated measurements of mobility dynamics (e.g. diffusion, transport) of fluorescent-labeled molecules in living cells.

Performance and use

Spectra change of Dronpa

The absorption spectrum of Dronpa at pH7.4 displays a major peak at 503 nm and a minor peak at 390 nm. The absorbance of Dronpa at pH 7.4 were measured during continuous illumination at 490 nm ± 10. After a 40-min incubation, nearly all the Dronpa molecules had been converted into a neutral, nonfluorescent state.

Ando, R., Mizuno, H. and Miyawaki, A.(2004) Science 306, 1370-1373.

Photochoromic properties of Dronpa

Dronpa was transfected into HeLa cells followed by fixation and the time course of fluorescence was monitored. Photoactivation of DG1 required much less photon energy than did photobleaching, with respective quantum yields of 0.37 (ΦPA) and 0.00032 (ΦPB).

Ando, R., Mizuno, H. and Miyawaki, A.(2004) Science 306, 1370-1373.

Monitoring the nuclear import of ERK1-Dronpa in Cos7 cells

(A) Confocal image of a COS7 cell expressing ERK1-Dronpa. (B) Schematic of the cell shown in A. The regions illuminated with the 405-nm laser line are shown in gray. The illuminated regions were　inside the cytosol (left) or nucleus (right) to monitor nuclear import (C→N). The time periods monitored for nuclear import are shown in red. (D and E) Nuclear import of ERK1-Dronpa before stimulation of D or after an 11-min incubation with EGF (E).

Ando, R., Mizuno, H. and Miyawaki, A.(2004) Science 306, 1370-1373.

Molecular imaging of Sar1P-Dronpa in yeast

CoralHue® Dronpa-Green1 is useful for monitoring molecular dynamics. Sar1P, of a small G protein family, was fused with Dronpa-Green1 (Sar1P-Dronpa). Sar1P-Dronpa was expressed in yeast. The videos show Sar1P-Dronpa monitoring molecular dynamics in the mother cell and daughter cells.

Provided by Dr. Kurokawa and Dr. Nakano Live Cell Molecular Imaging Research Team, ASI, RIKEN

Citations

  1. Ando R, Mizuno H, Miyawaki A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science. (2004) 306: 1370-3. PMID: 15550670
  2. Ando R, Flors C, Mizuno H, Hofkens J, Miyawaki A. Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants. Biophys J. (2007) 92:L97-9. PMID: 17384059
  3. Habuchi S, Ando R, Dedecker P, Verheijen W, Mizuno H, Miyawaki A, Hofkens J. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci U S A. (2005) 102:9511-6. PMID: 15972810
  4. Habuchi S, Dedecker P, Hotta J, Flors C, Ando R, Mizuno H, Miyawaki A, Hofkens J. Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitchingPhotochem Photobiol Sci. (2006) 5:567-76. PMID: 16761085

Fluorescent properties

Dronpa-Green spectrum data files (text files)

Note: The file is in a tab-delimited text format. It contains values of the wavelength (0.5nm spacing) and brightness (fluorescence intensity peak value normalized to 1). Use a spreadsheet program to create a spectrum that will help you in choosing the appropriate excitation filter, dichroic mirror and fluorescence filter.

*1Brightness: Molar Extinction Coefficient ×Fluorescence Quantum Yield / 1000 *2Toxicity when expressed in HeLa cells

Recommended antibodies

CoralHue®Dronpa-Green1 can be recognized using antibodies as shown below.

  1. Anti-Dronpa for IP, Code No. M118-3M

WB: Western blotting, IP: Immunoprecipitation