Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm

Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent p...

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Bibliographic Details
Published in:Scientific reports 2016-01, Vol.6 (1), p.18459-18459, Article 18459
Main Authors: El Khatib, Mariam, Martins, Alexandre, Bourgeois, Dominique, Colletier, Jacques-Philippe, Adam, Virgile
Format: Article
Language:English
Subjects:
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Biochemistry, Molecular Biology
E coli
Escherichia coli - genetics
Escherichia coli - metabolism
Gene Expression
Green fluorescent protein
Humanities and Social Sciences
Hydrogen Bonding
Life Sciences
Luminescent Proteins - chemistry
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Microscopy, Fluorescence
Models, Molecular
Molecular Imaging
multidisciplinary
Mutation
Periplasm
Periplasm - metabolism
Protein Conformation
Proteins
Quantitative Structure-Activity Relationship
Science
Structural Biology
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Summary:Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2 and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis - trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep18459