Real-time monitoring of glutathione in living cells using genetically encoded FRET-based ratiometric nanosensor

Mohammad Ahmad,Ambreen Asif,Altaf Ahmad,Naser A. Anjum

doi:10.1038/s41598-020-57654-y

Mohammad Ahmad, Ambreen Asif + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-57654-y

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Journal: Scientific Reports	Publication Date: Jan 22, 2020
Citations: 15	License type: open-access

Affiliation: Jamia Hamdard, Aligarh Muslim University

Abstract

Reduced glutathione (GSH) level inside the cell is a critical determinant for cell viability. The level of GSH varies across the cells, tissues and environmental conditions. However, our current understanding of physiological and pathological GSH changes at high spatial and temporal resolution is limited due to non-availability of practicable GSH-detection methods. In order to measure GSH at real-time, a ratiometric genetically encoded nanosensor was developed using fluorescent proteins and fluorescence resonance energy transfer (FRET) approach. The construction of the sensor involved the introduction of GSH binding protein (YliB) as a sensory domain between cyan fluorescent protein (CFP; FRET donor) and yellow fluorescent protein (YFP; FRET acceptor). The developed sensor, named as FLIP-G (Fluorescence Indicator Protein for Glutathione) was able to measure the GSH level under in vitro and in vivo conditions. When the purified FLIP-G was titrated with different concentrations of GSH, the FRET ratio increased with increase in GSH-concentration. The sensor was found to be specific for GSH and also stable to changes in pH. Moreover, in live bacterial cells, the constructed sensor enabled the real-time quantification of cytosolic GSH that is controlled by the oxidative stress level. When expressed in yeast cells, FRET ratio increased with the external supply of GSH to living cells. Therefore, as a valuable tool, the developed FLIP-G can monitor GSH level in living cells and also help in gaining new insights into GSH metabolism.

Highlights

Reduced glutathione (GSH) level inside the cell is a critical determinant for cell viability
Green fluorescent protein (GFP) variants and ligand sensing domains have been used to design genetically encoded sensors based on fluorescence resonance energy transfer (FRET)
It was argued that the conformational change in bound and unbound state of YliB would lead to a change in FRET efficiency between the fluorophores

Summary

Introduction

Reduced glutathione (GSH) level inside the cell is a critical determinant for cell viability. In order to measure GSH at real-time, a ratiometric genetically encoded nanosensor was developed using fluorescent proteins and fluorescence resonance energy transfer (FRET) approach. The developed sensor, named as FLIP-G (Fluorescence Indicator Protein for Glutathione) was able to measure the GSH level under in vitro and in vivo conditions. In vivo oxidative stress level can be monitored through the measurement of intracellular GSH concentration of the cells[2,4,6,7] Techniques such as fluorimetry, bioluminometery, HPLC and LC/MS have so far been used to detect and quantify different forms of GSH in biological samples[8]. The conformational change in the sensory domain is involved in the detection of the target metabolite through the genetically encoded FRET-based sensor (a tandemly fused protein). The efficacy of drugs in patients with chronic myeloid leukemia (CML) has been measured through FRET-based biosensor-mediated monitoring of the activity of BCR-ABL kinase that is causatively expressed in CML patients[20]

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