Structural studies of intracellular disulfide bonding as a novel stabilizing mechanism in proteins from hyperthermophilic microbes

This work presents the computational and experimental studies that led to the discovery of widespread disulfide bond abundance in cytosolic proteins of certain thermophilic and hyperthermophilic microorganisms. Disulfide bonds have long been recognized as stabilizing elements in protein folding. However, the observation that structurally stabilizing disulfide bonds occur almost exclusively in extracytosolic proteins led to the belief that the reducing environment of the cytosol does not permit disulfide bond formation. This assumption has recently been called into question, as the discovery of three disulfide bonds in the crystal structure of a hyperthermophilic adenylosuccinate lyase raised the possibility that certain thermophilic organisms may utilize disulfide bond formation in cytosolic proteins as a stabilizing mechanism against thermal denaturation. The results of our computational analysis of thermophilic and hyperthermophilic genomes indicate that disulfide bonds are common in certain thermophiles, with an apparent correlation between the extent of disulfide bonding and the optimal temperature of growth. Furthermore, fluorescent labeling studies and 2D diagonal gel electrophoresis of the hyperthermophilic archaeon Pyrobaculum aerophilum confirm the widespread utilization of disulfide bonds in cytosolic proteins, and demonstrate that many protein-protein complexes are stabilized by the formation of intermolecular disulfide bonds between the subunits. Finally, the 1.60A crystal structure of the P. aerophilum citrate synthase homodimer, identified from 2D diagonal gel electrophoresis as a disulfide-bonded protein complex, reveals that the dimer forms a protein catenane, with subunit chains cyclized and interlinked by disulfide bond formation. Thermal denaturation studies demonstrate the stabilizing effect of this unusual topological feature.