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A GFP-based ratiometric sensor for cellular methionine oxidation. | LitMetric

AI Article Synopsis

  • Methionine oxidation is a reversible change that affects protein function and is linked to aging and diseases, but measuring it in living cells has been challenging.
  • The authors developed a new genetically encoded probe called GEPMO, which uses a modified version of green fluorescent protein to detect methionine oxidation through changes in fluorescence.
  • This sensor can be used in various experiments, including live-cell imaging and fluorescence-activated cell sorting, and can provide detailed information about methionine oxidation in different parts of the cell.

Article Abstract

Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation. Placed at amino-acid position 147 in an otherwise methionine-less sfGFP, the oxidation of this specific methionine to methionine sulfoxide results in a ratiometric fluorescence change when excited with ∼400 and ∼470 nm light. The strength and homogeneity of the sensor expression is suited for live-cell imaging as well as fluorescence-activated cell sorting (FACS) experiments using standard laser wavelengths (405/488 nm). Expressed in mammalian cells and also in S. cerevisiae, the sensor protein faithfully reports on the status of methionine oxidation in an integrating manner. Variants targeted to membranes and the mitochondria provide subcellular resolution of methionine oxidation, e.g. reporting on site-specific oxidation by illumination of endogenous protoporphyrin IX.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9552927PMC
http://dx.doi.org/10.1016/j.talanta.2022.123332DOI Listing

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