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Angew Chem Int Ed Engl. 2017 Sep 11;56(38):11628-11633. doi: 10.1002/anie.201706121. Epub 2017 Jul 11.

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion.

Author information

1
Department for Biosystems Science and Engineering (D-BSSE), Eidgenössische Technische Hochschule (ETH) Zurich, 4058, Basel, Switzerland.
2
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, 20147, USA.
3
Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany.
4
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), 76344, Eggenstein-Leopoldshafen, Germany.
5
Institute of Nanotechnology, Karlsruhe Institute of Technology (KIT), 76344, Eggenstein-Leopoldshafen, Germany.
6
Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Abstract

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.

KEYWORDS:

biophysics; fluorescent proteins; primed conversion; super-resolution microscopy; triplet states

PMID:
28661566
DOI:
10.1002/anie.201706121
[Indexed for MEDLINE]

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