Abstract
Simple SummarySpatially and temporally defined H2O2 signatures are essential parts of various signaling pathways. Therefore, monitoring H2O2 dynamics with high spatio–temporal resolution is significantly important to understand how this ubiquitous signaling molecule can control diverse cellular responses. In this study, we designed and characterized a Fluorescence Resonance Energy Transfer (FRET)-based genetically encoded H2O2 sensor that provides a powerful tool to monitor the spatio–temporal dynamics of H2O2 fluxes. We have used this sensor to monitor the flux of H2O2 in live cells under stress conditions. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively and would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.
Highlights
Reactive oxygen species (ROS) are short-lived and highly reactive molecules formed upon incomplete reduction in oxygen [1,2]
We developed a reliable tool, FLIP-H2 O2 that can measure the concentration of H2 O2 in a ratiometric manner and unlike the previously reported sensors, as their measurements are affected by auto-fluorescence [14], sensor protein concentrations, and excitation wavelength in lower visible region [16]
The development and adaptation of a Fluorescence Resonance Energy Transfer (FRET) nanosensor provides the capacity toto visualize changes in the transport and accumulation of metabolites and small molecules at the cellular level, which changes in the transport and accumulation of metabolites and small molecules at the cellular level, which required identify the regulatory switches the metabolic network
Summary
Reactive oxygen species (ROS) are short-lived and highly reactive molecules formed upon incomplete reduction in oxygen [1,2]. They are the key regulators of various biological processes like signaling and development in living organisms. H2 O2 has been observed as a specific component of numerous signaling pathways as well as a well-known toxic molecule among all the ROS present inside the cells [6]. Detection of ROS was achieved by using various fluorescent probes such as 20,70-dichlorofluorescein diacetate, dihydroethidium (DHE), dihydro-20,4,5,6,7,70-hexafluoro fluorescein but these probes are difficult to deliver into living cells and cause the toxicity [9]. Attachment of various fluorescent molecules with aryl boronates produces a fluorescent product when it reacts with H2 O2
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