Green-to-Red Photoconvertible Dronpa Mutant for Multimodal Super-resolution Fluorescence Microscopy

Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent pr...

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Bibliographic Details
Published in:ACS nano Vol. 8; no. 2; pp. 1664 - 1673
Main Authors: Moeyaert, Benjamien, Nguyen Bich, Ngan, De Zitter, Elke, Rocha, Susana, Clays, Koen, Mizuno, Hideaki, van Meervelt, Luc, Hofkens, Johan, Dedecker, Peter
Format: Journal Article
Language:English
Published: United States American Chemical Society 25.02.2014
Subjects:
Biochemistry, Molecular Biology
Biophysics
Displays
Fluctuation
Fluorescence
Imaging
Life Sciences
Microscopy
Nanostructure
Optimization
Proteins
Strategy
Structural Biology
fluorescent proteins
protein engineering
PALM
Dronpa
pcSOFI
crystal structure determination
super-resolution fluorescence microscopy
ISSN:1936-0851, 1936-086X, 1936-086X
Online Access:Get full text
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Summary:Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.
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ISSN:1936-0851
1936-086X
1936-086X
DOI:10.1021/nn4060144