Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Redox-sensitive probes are crucial for monitoring cellular oxidative stress.
• Existing probes often lack sufficient dynamic range or optimal redox potential for specific cellular compartments.
• Förster resonance energy transfer (FRET) based probes offer a sensitive mechanism for detecting redox changes.

Purpose of the Study:

• To optimize a novel green fluorescent protein (GFP)-based redox-sensitive probe with improved dynamic range and a well-positioned oxidative midpoint redox potential.
• To characterize the factors contributing to enhanced FRET performance in the optimized probe.
• To determine the suitability of the optimized probe for measuring redox potentials in mammalian cell oxidative compartments.

Main Methods:

• Engineering of an α-helical linker (RL7) with extended length to modulate FRET efficiency.
• Site-directed mutagenesis of cysteine residues within the linker to identify key residues for disulfide bond formation.
• Quantitative comparison and exchange of various cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) FRET pairs, including monomeric variants, attached to the RL7 linker.
• In vitro characterization of FRET efficiency changes upon redox state transition.
• Determination of the probe's midpoint redox potential.

Main Results:

• Extension of the α-helical linker and optimization of cysteine residues led to decreased FRET in the reduced state and increased FRET in the oxidized state, significantly enhancing the dynamic range.
• Monomeric fluorescent protein variants (mCyPet, mYPet) prevented heterodimerization and restored redox sensitivity, unlike tandem CFP/YFP pairs.
• The optimized probe (ECFP-RL7-EYFP) demonstrated a six-fold increase in FRET efficiency from oxidized to reduced states.
• The probe's midpoint redox potential was determined to be -143 ± 6 mV, suitable for measuring glutathione redox potential in cellular oxidative compartments.

Conclusions:

• The optimized redox-sensitive probe exhibits significantly enhanced dynamic range and a favorable midpoint redox potential for cellular applications.
• The design strategy involving linker engineering and the use of monomeric fluorescent proteins is effective for creating robust redox sensors.
• This novel probe is well-suited for accurately measuring glutathione redox potentials in specific oxidative compartments of mammalian cells, such as the endoplasmic reticulum.