Monitoring of methionine oxidation with fluorescent proteins

Methionine (Met) oxidation to methionine sulfoxide (MetO) is a post-translational protein modification that can appear unspecifically during exposure of Met to oxidants in normal growth and oxidative stress, such as occurring in aged organisms and in various degenerative diseases and pathologies. The importance of MetO is emphasized by a set of methionine sulfoxide reductases (Msrs) present in virtually almost all organisms. Studies of Met oxidation typically involve biochemical work-up, thus precluding real-time measurements in living cells. Moreover, there is no specific antibody against MetO for the convenient labeling or enrichment of oxidized proteins. Therefore, there is a demand for a tool that can report Met oxidation in vitro and in living cells. Here we introduce a Genetically-Encoded fluorescent Probe of Met Oxidation (GEPMO). We designed and optimized variants of superfolder green fluorescent protein (sfGFP) which provide excitation-ratiometric fluorescent signals reporting on the degree of Met oxidation. Produced in a bacterial expression system, the variants of sfGFP were characterized under fully controlled conditions in a spectrofluorometer. A derivative with the best performance was chosen for further work. Oxidation of the principal solvent-exposed Met147 in GEPMO changed the distribution of chromophores neutral and protonated states which can be monitored as the ratio of fluorescence intensities F400 / F470. Incubation of the indicator with Met oxidants showed robust changes in the fluorescent ratio F400 / F470; full oxidation resulted in an about 2-fold decrease. Mass spectrometry analysis confirmed that oxidation of a Met residue in the indicator is responsible for the ratio changes. We determined the rate constant of Met oxidation with chloramine T. We demonstrated that chloramine T is a Met-specific oxidant. GEPMO fills a gap in the genetically encoded redox sensor toolkit, which is currently represented only by Cys-based sensors.