Principal Investigator Ronald Raines
Making correct disulfide linkages between cysteine residues (and undoing incorrect ones) is a critical but often complex aspect of protein folding. We addressed this problem from several directions using diverse tools. Protein disulfide isomerase (PDI) and thioredoxin (Trx) are enzymes that catalyze disulfide-bond formation with a common active-site motif: Cys–Xaa–Xaa–Cys, where Xaa can vary. We are combining in vitro mechanistic studies of these two enzymes with molecular genetics to define the optimal attributes of a folding catalyst, which appear to be a disulfide bond of high reduction potential (E°′) within the active site and a solvent-exposed cysteine of low pKa. Inspired by PDI and Trx, we are creating small-molecule dithiols with low thiol pKa and high disulfide reduction potential that are efficacious organocatalysts of oxidative protein folding, both in vitro and in cellulo. Finally, we designed and synthesized dithiobutylamine (DTBA; Sigma–Aldrich 774405), which is superior to the venerable dithiothreitol (DTT) as a biochemical reducing agent. We are now using DTBA conjugates to deliver electrons into physiological systems with surgical precision.