Direct structural evidence of protein redox regulation obtained by in-cell NMR

Biochim Biophys Acta. 2016 Feb;1863(2):198-204. doi: 10.1016/j.bbamcr.2015.11.009. Epub 2015 Nov 14.

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.

Keywords: Disulfide bond; Glutathione; In-cell NMR; Nuclear magnetic resonance; Redox regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Blotting, Western
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cellular Microenvironment
  • Cytoplasm / metabolism
  • Escherichia coli / cytology
  • Escherichia coli / metabolism
  • Glutathione / metabolism
  • Glutathione Disulfide / metabolism
  • HEK293 Cells
  • Humans
  • Magnetic Resonance Spectroscopy / methods*
  • Mitochondrial Membrane Transport Proteins / genetics
  • Mitochondrial Membrane Transport Proteins / metabolism*
  • Oxidation-Reduction
  • Sulfhydryl Compounds / metabolism
  • Superoxide Dismutase / genetics
  • Superoxide Dismutase / metabolism*
  • Superoxide Dismutase-1

Substances

  • COX17 protein, human
  • Carrier Proteins
  • MIA40 protein, human
  • Mitochondrial Membrane Transport Proteins
  • SOD1 protein, human
  • Sulfhydryl Compounds
  • Superoxide Dismutase
  • Superoxide Dismutase-1
  • Glutathione
  • Glutathione Disulfide