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Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light


Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes1,2. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed3. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP5, Kaede6, EosFP7, PS-CFP8, Dronpa9, PA-mRFP110 and KikGR11 require light in the UV-violet spectral region for activation through one-photon excitation—such light can be phototoxic to some biological systems12. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 °C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.

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Figure 1: Labeling of intracellular oligomerizing proteins with Dendra.
Figure 2: Tracking Dendra-fibrillarin in HeLa cells.
Figure 3: Tracking Dendra-vimentin in a HeLa cell containing vimentin in both dots and filaments.

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We thank Robert Goldman for providing us with the EGFP-vimentin vector and Alexey V. Feofanov for help in light intensity measurements. This work was supported by grants from the European Commission FP-6 Integrated Project LSHG-CT-2003-503259 (K.A.L.), the Russian Academy of Sciences for the program Molecular and Cell Biology (S.L.) and by grants GM070358 and DA019980 from the National Institutes of Health (V.V.V.).

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Correspondence to Konstantin A Lukyanov.

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D.B.S. and T.V.C. are employees of Evrogen, which is developing this technology for commercial application.

Supplementary information

Supplementary Fig. 1

Amino acid sequence alignment of Dendra, monomeric Azami Green (mAG), Kaede, monomeric EosFP (mEosFP), KikGR and GFP. (PDF 28 kb)

Supplementary Fig. 2

Gel-filtration analysis of wild type dendGFP (blue circles), and its mutants V3 (green squares) and Dendra (red triangles). (PDF 22 kb)

Supplementary Fig. 3

Dendra spectral properties. (PDF 22 kb)

Supplementary Table 1

Spectral properties of wild type dendGFP and its monomeric mutant Dendra. (PDF 32 kb)

Supplementary Table 2

Some properties of known photoactivatable proteins. (PDF 65 kb)

Supplementary Video 1

Dendra-fibrillarin fusion movement tracking in HeLa cell nucleus. (MOV 799 kb)

Supplementary Video 2

Dendra-vimentin fusion movement tracking in HeLa cell. (MOV 731 kb)

Supplementary Data (PDF 23 kb)

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Gurskaya, N., Verkhusha, V., Shcheglov, A. et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24, 461–465 (2006).

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