SEMI-SYNTHESIS AND OXIDATIVE FOLDING STUDIES OF SELENO-ONCONASE

The sulfur of cysteine (Cys) and selenium of its natural analog selenocysteine (Sec) share many properties, yet they differ in some crucial properties, making selenium a better nucleophile and electrophile in thiol-disulfide-like exchange reactions. Since the formation of disulfide bonds is crucial for the emergence of the protein 3-D structures, the replacement of Cys by Sec can lead to drastic changes in the folding kinetics of the proteins. Past researches have shown that Sec substitution into proteins and polypeptides facilitates folding processes and may steer their folding into more predictive and productive routes.

This approach is still to be examined in other protein models – such as Onconase. Onconase belongs to RNase A superfamily, possessing similar structure and function. It was shown that Onconase exhibits a cytotoxic effect towards human cancer cells and it has reached phase III clinical trials. Onconase is composed of 104 residues and is stabilized by four disulfide bonds, compared to the usual three in RNase A family. The oxidative folding mechanism of Onconase has been thoroughly studied, pointing to a pathway including two highly heterogeneous intermediates, and three homogeneous intermediates which lack at least one of the two disulfide bonds that are the last formed in the native state.

This project is aimed at broadening the scope of selenium folding enhancements to semi-synthetic proteins. Using Intein and native chemical ligation, two analogs of Onconase with a single Sec substitution will be prepared. The analog proteins will be subject to in vitro oxidative folding and activity studies, to compare the folding pathway and rate while ensuring that the wildtype activity is maintained.