Studies on the unfolding and refolding of multi-subunit proteins

Abstract

Studies on the unfolding and refolding of multi-subunit proteins have been carried out and the results obtained for translocated proteins have been compared where possible with those obtained for non-translocated proteins. This comparison should indicate the way in which translocation affects the folding and assembly of such proteins. The enzymes studied were the glutamate dehydrogenases from bovine liver, baker's yeast and Clostridium symbiosum: the cytoplasmic and mitochondrial aspartate aminotransferase isoenzymes from pig heart and citrate synthase from pig heart. The unfolding of the enzymes by guanidinium chloride (GdnHCl) was studied by monitoring loss of catalytic activity and changes in structure by fluorescence, circular dichroism and exposure of reactive thiol groups. It was found that loss of enzyme activity occurs at lower concentrations of GdnHCl than any major changes in the structure. The refolding of the enzymes was studied by measuring the regain of catalytic activity on dilution of the GdnHCl. It was found that only the cytoplasmic aspartate aminotransferase enzyme was able to regain activity from the unfolded state. In comparison, the translocated proteins studied bovine liver glutamate dehydrogenase, mitochondrial aspartate aminotransferase and citrate synthase could not be refolded after denaturation. This inability of translocated proteins to refold from their isolated, unfolded subunits implies that other factors are involved i the folding and assembly of translocated proteins in vivo. It is possible that chaperone proteins nay be involved in this process.