Oxidative Folding in Bacteria

Oxidative folding, the process by which folding and disulfide oxidation occur in concert, is a critical step in the production of many extracellular proteins and is therefore centrally linked to a vast multitude of important physiological functions. The primary focus of this dissertation is the remarkable disulfide oxidoreductase DsbA, the sole catalyst of oxidative folding in Escherichia coli. DsbA was the first oxidative folding catalyst to be discovered, and remains the strongest known oxidant among the thioredoxin superfamily of disulfide oxidoreductases due to unique biochemical and biophysical properties. Through the activity of its substrate repertoire, which includes adhesion structures and toxins, DsbA is an essential component of many pathogenic processes and therefore is an active target for the development of novel antibiotics. Though DsbA has been analyzed through a host of biochemical, genetic, and cellular experiments over the quarter-century since its identification, the elucidation of certain mechanistic details of its catalytic process have proven elusive to conventional techniques. This primarily results from the experimental difficulties in independently monitoring the progress of folding and oxidation during oxidative folding that arise with conventional, ensemble-averaged approaches. In this work, single molecule force spectroscopy methods are applied to investigate the process of oxidative folding as catalyzed by DsbA. Through observing single substrate molecules as they undergo DsbA-catalyzed oxidative folding, a precise kinetic analysis of the enzyme is constructed. DsbA is demonstrated to be a highly effective catalyst of oxidative folding, outperforming its eukaryotic counterpart by substantial margins in every metric considered. This efficacy complements the strong preference for simpler disulfide connectivity patterns in the Escherichia coli proteome, which in conjunction likely represent a strategy for navigating the physiological demands that are imposed by the inherent speed of prokaryotic life, in which a generation can be as short as twenty minutes.