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

Authors:
Dr. Virgile Adam, PhD
Dr. Virgile Adam, PhD
CNRS / Institute for Structural Biology (IBS)
Research scientist
single molecule fluorescence microscopy, fluorescent protein engineering, X-ray crystallography
Grenoble | France

Sci Rep 2016 Jan 6;6:18459. Epub 2016 Jan 6.

Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.

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.

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http://dx.doi.org/10.1038/srep18459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702087PMC
January 2016
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