Dataset Information

A Forster Resonance Energy Transfer-Based Ratiometric Sensor with the Allosteric Transcription Factor TetR.

ABSTRACT: A recent description of an antibody-free assay is significantly extended for small molecule analytes using allosteric transcription factors (aTFs) and Förster resonance energy transfer (FRET). The FRET signal indicates the differential binding of an aTF-DNA pair with a dose-dependent response to its effector molecule, i.e., the analyte. The new sensors described here, based on the well-characterized aTF TetR, demonstrate several new features of the assay approach: 1) the generalizability of the sensors to additional aTF-DNA-analyte systems, 2) sensitivity modulation through the choice of donor fluorophore (quantum dots or fluorescent proteins, FPs), and 3) sensor tuning using aTF variants with differing aTF-DNA binding affinities. While all of these modular sensors self-assemble, the design reported here based on a recombinant aTF-FP chimera with commercially available dye-labeled DNA uses readily accessible sensor components to facilitate easy adoption of the sensing approach by the broader community.

SUBMITTER: Nguyen TT

PROVIDER: S-EPMC7359203 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A Förster Resonance Energy Transfer-Based Ratiometric Sensor with the Allosteric Transcription Factor TetR.

Nguyen Thuy T TT Chern Margaret M Baer R C RC Galagan James J Dennis Allison M AM

Small (Weinheim an der Bergstrasse, Germany) 20200406 17

A recent description of an antibody-free assay is significantly extended for small molecule analytes using allosteric transcription factors (aTFs) and Förster resonance energy transfer (FRET). The FRET signal indicates the differential binding of an aTF-DNA pair with a dose-dependent response to its effector molecule, i.e., the analyte. The new sensors described here, based on the well-characterized aTF TetR, demonstrate several new features of the assay approach: 1) the generalizability of the ...[more]

PMID: 32249506

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Project description:The glutathione thiol/disulfide couple is the major redox buffer in the endoplasmic reticulum (ER); however, mechanisms by which it contributes to the tightly regulated redox environment of this intracellular organelle are poorly understood. The recent development of genetically encoded, ratiometric, single green fluorescent protein-based redox-sensitive (roGFP) sensors adjusted for more oxidative environments enables non-invasive measurement of the ER redox environment in living cells. In turn, Förster resonance energy transfer (FRET) sensors based on two fluorophore probes represent an alternative strategy for ratiometric signal acquisition. In previous work, we described the FRET-based redox sensor CY-RL7 with a relatively high midpoint redox potential of -143 mV, which is required for monitoring glutathione potentials in the comparatively high oxidative environment of the ER. Here, the efficacy of the CY-RL7 probe was ascertained in the cytosol and ER of live cells with fluorescence microscopy and flow cytometry. The sensor was found to be fully reduced at steady state in the cytosol and became fully oxidized in response to treatment with 1-chloro-2,4-dinitrobenzene, a depletor of reduced glutathione (GSH). In contrast, the probe was strongly oxidized (88%) upon expression in the ER of cultured cells. We also examined the responsiveness of the ER sensor to perturbations in cellular glutathione homeostasis. We observed that the reductive level of the FRET sensor was increased two-fold to about 28% in cells pretreated with N-acetylcysteine, a substrate for GSH synthesis. Finally, we evaluated the responsiveness of CY-RL7 and roGFP1-iL to various perturbations of cellular glutathione homeostasis to address the divergence in the specificity of these two probes. Together, the present data generated with genetically encoded green fluorescent protein (GFP)-based glutathione probes highlight the complexity of the ER redox environment and indicate that the ER glutathione pool may be more oxidized than is currently considered.

Dataset Information

A Forster Resonance Energy Transfer-Based Ratiometric Sensor with the Allosteric Transcription Factor TetR.

Publications

A Förster Resonance Energy Transfer-Based Ratiometric Sensor with the Allosteric Transcription Factor TetR.

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