Novel reversibly switchable fluorescent proteins for RESOLFT and STED nanoscopy engineered from the bacterial photoreceptor YtvA

The reversibly switchable fluorescent proteins (RSFPs) commonly used for RESOLFT nanoscopy have been developed from fluorescent proteins of the GFP superfamily. These proteins are bright, but exhibit several drawbacks such as relatively large size, oxygen-dependence, sensitivity to low pH, and limit...

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Bibliographic Details
Published in:Scientific reports 2018-02, Vol.8 (1), p.2724-9, Article 2724
Main Authors: Gregor, Carola, Sidenstein, Sven C., Andresen, Martin, Sahl, Steffen J., Danzl, Johann G., Hell, Stefan W.
Format: Article
Language:English
Subjects:
14/19
14/35
14/5
14/63
631/1647/245/2225
631/1647/328/2238
Flavin mononucleotide
Green fluorescent protein
Humanities and Social Sciences
multidisciplinary
pH effects
Photoreceptors
Proteins
Science
Science (multidisciplinary)
Ultraviolet radiation
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Summary:The reversibly switchable fluorescent proteins (RSFPs) commonly used for RESOLFT nanoscopy have been developed from fluorescent proteins of the GFP superfamily. These proteins are bright, but exhibit several drawbacks such as relatively large size, oxygen-dependence, sensitivity to low pH, and limited switching speed. Therefore, RSFPs from other origins with improved properties need to be explored. Here, we report the development of two RSFPs based on the LOV domain of the photoreceptor protein YtvA from Bacillus subtilis . LOV domains obtain their fluorescence by association with the abundant cellular cofactor flavin mononucleotide (FMN). Under illumination with blue and ultraviolet light, they undergo a photocycle, making these proteins inherently photoswitchable. Our first improved variant, rsLOV1, can be used for RESOLFT imaging, whereas rsLOV2 proved useful for STED nanoscopy of living cells with a resolution of down to 50 nm. In addition to their smaller size compared to GFP-related proteins (17 kDa instead of 27 kDa) and their usability at low pH, rsLOV1 and rsLOV2 exhibit faster switching kinetics, switching on and off 3 times faster than rsEGFP2, the fastest-switching RSFP reported to date. Therefore, LOV-domain-based RSFPs have potential for applications where the switching speed of GFP-based proteins is limiting.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-018-19947-1