An NMR-based biosensor to measure stereo-specific methionine sulfoxide reductase (MSR) activitiesin vitroandin vivo

Abstract

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S-or R-diastereoisomers of methioninesulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments that range from native cell lysates to zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. Our approach further extends high-resolution incell NMR measurements of exogenously delivered biomolecules to an entire multicellular organism.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…The latter ones have been utilized only very recently. 277,278 We focus on the better established X. laevis oocytes below. These are quite appealing systems for in-cell NMR studies: i) only a ~170 of them are necessary to fill a 5 mm NMR tube; ii) they can be manipulated and microinjected cell by cell in about one hour; iii) they do not require a specific wet-lab, even though a source of oocytes has to be found (commercial companies sell them at ~1-2 $ a piece); iv) they can stand the injection of about 20-50 nL of highconcentration material, which generates ~20-50-fold intracellular dilutions; v) they can survive and maintain their integrity about 18 hours in the NMR tube without any medium replenishment; vi) they are very-well known model systems in biology.…”

“…730,731 15 N-labeled reporters of kinases and of methionine sulfoxide reductases have also been used in cells. 277,298…”