The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase

The oxidative refolding of hen lysozyme has been studied by a variety of ti me-resolved biophysical methods in conjunction with analysis of folding int ermediates using reverse-phase HPLC. in order to achieve this, refolding co nditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermedia tes with on average two disulfide bonds is formed rapidly from the fully re duced protein after initiation of folding. Following structural collapse, t he majority of molecules slowly form the four-disulfide-containing fully na tive protein via rearrangement of a highly native-like, kinetically trapped intermediate, des-[76-94], although a significant population (similar to 3 0%) appears to fold more quickly via other three-disulfide intermediates. T he folding catalyst PDI increases dramatically both yields and rates of lys ozyme refolding, largely by facilitating the conversion of des-[76-94] to t he native state. This suggests that acceleration of the folding rate mag be an important factor in avoiding aggregation in the intracellular environme nt.