A Highly Stable Engineered Disulfide Bond in the Dimer Interface of Orotate Phosphoribosyl Transferase Measures Cytosolic Redox Conditions in Yeast.

Although structural disulfides are very rarely found in cytoplasmic proteins, disulfides can form in the cytosol if they are stabilized sufficiently by the supporting protein structure. To investigate the redox properties of structural disulfide bonds, we introduced disulfide bonds into the cytosolic enzyme from , orotate phosphoribosyl transferase. Because this enzyme is a homodimer, the introduction of opposing cysteine residues (R44C and D92C) into separate monomers of the enzyme meant that disulfide bond formation could easily be followed by non-reducing SDS-PAGE. This disulfide bond was similar in strength to dithiothreitol, with a redox potential of -314 mV. Global thermostability of the disulfide-linked dimer increased by 5.9 °C relative to wild-type protein and 21.4 °C above its reduced form, without affecting the catalytic activity significantly. Combining an inactive subunit with the asymmetric nature of the disulfide bond enabled determination of the rates of subunit rearrangement and disulfide formation. The R44C/D92C double mutant resulted in the formation of a symmetric homodimer with two interchain disulfides, albeit without substantially increasing the stability relative to that of the dimer with the single disulfide bond. The engineered disulfide bonds are formed to a significant degree in yeast cytosol, revealing a redox potential of -300 mV. This is slightly lower than that previously determined using a GFP-based sensor, rxYFP, in yeast. Disulfide bond formation is particularly enhanced in mutants lacking glutathione reductase. Thus, the engineered disulfide provides an alternative method for determining the intracellular redox potential in living cells with an extended dynamic range relative to GFP-based sensors.