Small-molecule Catalysis of Native Disulfide Bond Formation in Proteins

Native disulfide bond formation is essential for the folding of many proteins. The enzyme protein disulfide isomerase (POI) catalyzes native disulfide bond formation using a Cys-Gly-HisCys active site. The active-site properties of POI (thiol pK. = 6.7 and disulfide E;O' = -180 mY) are critical for efficient catalysis. This Dissertation describes the design, synthesis, and characterization of small-molecule catalysts that mimic the active-site properties of POI. Chapter Two describes a small-molecule dithiol that has disulfide bond isomerization activity both in vitro and in vivo. The dithiol trans-I,2-bis(mercaptoacetamido)cyclohexane (BMC) has a thiol pKa value of 8.3 and a disulfide E;O' value of -240 mV.ln vitro, BMC increases the folding efficiency of disulfide--scrambled ribonuclease A (sRNase A). Addition of BMC to the growth medium of yeast cells causes an increase in the heterologous secretion of Schizosaccharomyces pombe acid phosphatase. which contains eight disulfide bonds. Chapter Three describes a tripeptide that is a functional mimic of POI. The disulfide bond stability of the peptide Cys-Gly-Cys-NH2 (E;O' = -167 m V) matches closely that of the POI active sites. In vitro, the reduced form of this peptide has higher disulfide bond isomerization activity than does the reduced form of BMC. The CGC sequence was incorporated into a protein scaffold via deletion of the proline residue from the CGPC active site of Escherichia coli thioredoxin (Trx). This deletion destabilizes the active-site disulfide bond ofTrx and confers substantial disulfide bond isomerization activity. Chapter Four describes the synthesis of an immobilized dithiol that is useful for protein folding in vitro. Tris(2-mercaptoacetamidoethyl)amine was synthesized and coupled to two different solid supports: NovaSyn~ TG Br resin (TentaGel resin, a co-polymer of styrene and bromine-functionalized polyethylene glycol) and styrene-glycidyl methacrylate microspheres. TentaGel presenting dithiol shows little disulfide bond isomerization activity with a sRNase A substrate, due to sterie inaccessibility of the dithiols within the interior of the TentaGel resin. In contrast, microspheres present dithiol with a high surface density and show disulfide bond isomerization activity that is both substantial and greater than microspheres presenting a monothiol. ii