Details

Autor(en) / Beteiligte

• Kuldyushev, Nikita
• Schönherr, Roland
• Coburger, Ina
• Ahmed, Marwa
• Hussein, Rama A.
• Wiesel, Eric
• Godbole, Amod
• Pfirrmann, Thorsten
• Hoshi, Toshinori
• Heinemann, Stefan H.

Titel

A GFP-based ratiometric sensor for cellular methionine oxidation

Ist Teil von

• Talanta (Oxford), 2022-06, Vol.243, p.123332-123332, Article 123332

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2022

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. [Display omitted] Highlights/Novelty:•GEPMO, a single-domain GFP-based genetically encoded probe for methionine oxidation, was constructed and optimized.•GEPMO yields specific and quantitative excitation ratiometric (≈400/470 nm) fluorescence signals reporting on the oxidation status of Met147.•GEPMO is suited for live-cell imaging and FACS analysis of mammalian cells and yeast, and targeted variants provide subcellular resolution.