title: Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide.
abstract: In cells, physiological and pathophysiological conditions may lead to the formation of methionine sulfoxide (MetO). This oxidative modification of methionine exists in the form of two diastereomers, R and S, and may occur in both free amino acid and proteins. MetO is reduced back to methionine by methionine sulfoxide reductases (MSRs). Methionine oxidation was thought to be a nonspecific modification affecting protein functions and methionine availability. However, recent findings suggest that cyclic methionine oxidation and reduction is a posttranslational modification that actively regulates protein function akin to redox regulation by cysteine oxidation and phosphorylation. Methionine oxidation is thus an important mechanism that could play out in various physiological contexts. However, detecting MetO generation and MSR functions remains challenging because of the lack of tools and reagents to detect and quantify this protein modification. We recently developed two genetically encoded diasterospecific fluorescent sensors, MetSOx and MetROx, to dynamically monitor MetO in living cells. Here, we provide a detailed procedure for their use in bacterial and mammalian cells using fluorimetric and fluorescent imaging approaches. This method can be adapted to dynamically monitor methionine oxidation in various cell types and under various conditions.
authors.affiliation: Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Zalán
authors.initials: Z
authors.lastname: Péterfi
authors.affiliation: Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France. Electronic address: lioneltarrago@msn.com.
authors.forename: Lionel
authors.initials: L
authors.lastname: Tarrago
authors.affiliation: Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address: vgladyshev@rics.bwh.harvard.edu.
authors.forename: Vadim N
authors.initials: VN
authors.lastname: Gladyshev
mesh_terms: D000818:Animals; D001419:Bacteria / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D008322:Mammals; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry* / Q000235:genetics; D057054:Molecular Imaging / Q000379:methods*; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational / Q000235:genetics; D013237:Stereoisomerism
chemical_list: D008715:Methionine; D051150:Methionine Sulfoxide Reductases; C013111:methionine sulfoxide
keywords: Methionine; Methionine sulfoxide; Sensors