title: Direct structural evidence of protein redox regulation obtained by in-cell NMR.
abstract: The redox properties of cellular environments are critical to many functional processes, and are strictly controlled in all living organisms. The glutathione-glutathione disulfide (GSH-GSSG) couple is the most abundant intracellular redox couple. A GSH redox potential can be calculated for each cellular compartment, which reflects the redox properties of that environment. This redox potential is often used to predict the redox state of a disulfide-containing protein, based on thermodynamic considerations. However, thiol-disulfide exchange reactions are often catalyzed by specific partners, and the distribution of the redox states of a protein may not correspond to the thermodynamic equilibrium with the GSH pool. Ideally, the protein redox state should be measured directly, bypassing the need to extrapolate from the GSH. Here, by in-cell NMR, we directly observe the redox state of three human proteins, Cox17, Mia40 and SOD1, in the cytoplasm of human and bacterial cells. We compare the observed distributions of redox states with those predicted by the GSH redox potential, and our results partially agree with the predictions. Discrepancies likely arise from the fact that the redox state of SOD1 is controlled by a specific partner, its copper chaperone (CCS), in a pathway which is not linked to the GSH redox potential. In principle, in-cell NMR allows determining whether redox proteins are at the equilibrium with GSH, or they are kinetically regulated. Such approach does not need assumptions on the redox potential of the environment, and provides a way to characterize each redox-regulating pathway separately.
authors.affiliation: Magnetic Resonance Center - CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Mercatelli
authors.affiliation: Magnetic Resonance Center - CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy; Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Letizia
authors.initials: L
authors.lastname: Barbieri
authors.affiliation: Magnetic Resonance Center - CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy; Department of Biomedical, Clinical and Experimental Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Luchinat
authors.affiliation: Magnetic Resonance Center - CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy; Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. Electronic address: banci@cerm.unifi.it.
authors.forename: Lucia
authors.initials: L
authors.lastname: Banci
mesh_terms: D000465:Algorithms; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D060833:Cellular Microenvironment; D000079922:Copper Transport Proteins; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1
chemical_list: C507282:CHCHD4 protein, human; C491006:COX17 protein, human; D002352:Carrier Proteins; D000079922:Copper Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C000606290:SOD1 protein, human; D013438:Sulfhydryl Compounds; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D005978:Glutathione; D019803:Glutathione Disulfide
keywords: Disulfide bond; Glutathione; In-cell NMR; Nuclear magnetic resonance; Redox regulation