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

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...

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Bibliographic Details
Published in:Angewandte Chemie 2017-09, Vol.129 (38), p.11786-11791
Main Authors: Mohr, Manuel Alexander, Kobitski, Andrei Yu, Sabater, Lluc Rullan, Nienhaus, Karin, Obara, Christopher John, Lippincott‐Schwartz, Jennifer, Nienhaus, Gerd Ulrich, Pantazis, Periklis
Format: Article
Language:English
Subjects:
Biophysik
Chemistry
Color
Conversion
Engineering
Fluorescence
Fluoreszierende Proteine
I.R. radiation
Illumination
Infrared radiation
Knowledge management
Localization
Microscopy
Position (location)
Primed Conversion
Proteins
Serine
Superauflösende Mikroskopie
Threonine
Triplett-Zustände
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Summary: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. Die Aufklärung des Mechanismus der Photokonvertierung fluoreszierender Proteine mittels blauem und nah‐infrarotem Licht (Primed Conversion) ermöglicht die Entwicklung photokonvertierbarer (pr‐) fluoreszierender Proteine. Ein pr‐Eos‐ und ein Wildtyp‐Eos‐Protein wurden gemeinsam genutzt für optische Nanoskopie (photoaktivierte Lokalisierungsmikroskopie) in zwei spektralen Kanälen.
ISSN:0044-8249
1521-3757
DOI:10.1002/ange.201706121