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ROS Live Cell Imaging During Neuronal Development
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Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments.

Vladimir L Kolossov1, Bryan Q Spring, Robert M Clegg

  • 1Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. viadimer@illinois.edu

Experimental Biology and Medicine (Maywood, N.J.)
|May 25, 2011
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Summary

This study optimized a novel green fluorescent protein (GFP)-based redox sensor, enhancing its dynamic range and redox potential for accurate cellular measurements. The improved probe offers a six-fold increase in Förster resonance energy transfer (FRET) efficiency.

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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.