Oxidative Protein Folding of Modified Disulfide-Rich Proteins

The in vitro oxidative folding has been extensively studied in the last four decades especially with small disulfide-rich proteins. One conclusion that these studies provide is that many disulfide-rich proteins follow two different folding model mechanisms; those seen for bovine pancreatic trypsin inhibitor (BPTI) and for hirudin, or a combination of the two folding models.
Selenocysteine (Sec) incorporation has been successfully used in protein folding studies including that of BPTI. Sec`s low redox potential, and pKa, as well as its increased nucleophilicity and electrophilicity can enhance thiol-disulfide-like exchange reactions that are essential for protein folding. Based on these results we wish to study the effect of Sec substitution on the folding of hirudin. Wild-type hirudin and its seleno-analogs containing one or two substitutions at different positions that could form native diselenide bonds have been prepared using solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). Moreover, we aim to shed more light on BPTI folding mechanism by using different strategy. BPTI folding mechanism was extensively studied, in which the reduced protein folds via bifurcated pathway. Roughly half of the reduced BPTI reach the native state via two intermediates: (N’) that lacks the [5-55] disulfide and N* that lacks the [30-51]. Formation of the fully oxidized BPTI requires partially unfolding that reduces the solvent exposed disulfide bridge [14-38] and rearrangement of theses intermediates. Therefore, we propose that replacing the [14-38] disulfide bond with highly stable thioacetal, will cause the protein to be trapped at the two stable intermediates N’ and N*, preventing the formation of the native form (N). This experiment will shed more light, and answer any further dispute about the folding mechanism of BPTI. Here we show preliminary data along these lines.