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 International Edition 2017-09, Vol.56 (38), p.11628-11633
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:
biophysics
Color
Conversion
Engineering
Fluorescence
fluorescent proteins
I.R. radiation
Illumination
Infrared radiation
Knowledge management
Localization
Luminescent Proteins - chemistry
Microscopy
Microscopy, Fluorescence
Photochemical Processes
Position (location)
primed conversion
Protein Engineering
Proteins
Serine
super-resolution microscopy
Threonine
triplet states
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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. Uncovering the mechanism of primed conversion allows for rational engineering of primed conversion capable (pr‐) fluorescent proteins. An engineered pr‐Eos and a wild‐type Eos protein are combined for dual‐color fluorescence nanoscopy using photoactivated localization microscopy.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201706121