Thiol chemistry in proteins
We have developed a simple assay to monitor the reduction, oxidation and isomerization of disulfide bonds in single proteins. Disulfide bond reactions are crucial to the function of one third of all proteins, yet these reactions remain poorly understood. We use proteins with engineered disulfide bonds to arrest the mechanical extension that occurs during mechanical unfolding (figure). Reduction of the disulfide bond after unfolding results into a further extension that is easily detectable with force spectroscopy. The reverse reaction (oxidative folding) is also easily studied by applying the force-quench protocol in the presence of a chaperone such as Protein Disulfide Isomerase (PDI), as we describe in our 2012 paper in Cell. Now, we are expanding our studies to DsbA, the main catalyst of oxidative folding in bacteria, to investigate oxidative folding of bacterial virulence factors. Using our cutting edge single-molecule techniques, we have also observed that thiol chemistry is altered in mutants of superoxide dismutase that cause familial Amyotrophic Lateral Sclerosis (Lou Gehrig's disease).