Single-fluorophore biosensors based on conformation-sensitive GFP variants

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
Agnès BonnotBertrand Lambolez

Abstract

The β-strands of GFP form a rigid barrel that protects the chromophore from external influence. Herein, we identified specific mutations in β-strand 7 that render the chromophore sensitive to interactions of GFP with another protein domain. In the process of converting the FRET-based protein kinase A (PKA) sensor AKAR2 into a single-wavelength PKA sensor containing a GFP and a quencher, we discovered that the quencher was not required and that the sensor response relied on changes in GFP intrinsic fluorescence. The identified mutations in β-strand 7 render GFP fluorescence intensity and lifetime sensitive to conformational changes of the PKA-sensing domain. In addition, sensors engineered from the GCaMP2 calcium indicator to incorporate a conformation-sensitive GFP (csGFP) exhibited calcium-dependent fluorescence changes. We further demonstrate that single GFP sensors report PKA dynamics in dendritic spines of neurons from brain slices on 2-photon imaging with a high signal-to-baseline ratio and minimal photobleaching. The susceptibility of GFP variants to dynamic interactions with other protein domains provides a new approach to generate single wavelength biosensors for high-resolution imaging.

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Citations

Oct 27, 2015·Sensors·Bernhard HochreiterJohannes A Schmid
Oct 7, 2015·Frontiers in Pharmacology·Valeriy M ParamonovAdolfo Rivero-Müller
Dec 30, 2014·Frontiers in Cellular Neuroscience·Kirill Gorshkov, Jin Zhang
Sep 6, 2014·Frontiers in Cellular Neuroscience·Shinobu NomuraRégine Hepp
Jul 12, 2017·Molecular Psychiatry·N BenamerL Tricoire
Sep 2, 2017·Basic Research in Cardiology·Charlotte D KoopmanTeun P de Boer
Aug 3, 2021·Journal of Neuroscience Methods·Crystian I MassengillHaining Zhong

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