Oxidative refolding of amyloidogenic variants of human lysozyme.

The oxidative refolding of human lysozyme and its two best characterised amyloidogenic variants, Ile56Thr and Asp67His, has been investigated in vitro by means of the concerted application of a range of biophysical techniques. The results show that in each case the ensemble of reduced denatured conformers initially collapses into a large number of unstructured intermediates with one or two disulphide bonds, the majority of which then fold to form the native-like three-disulphide intermediate, des-[77-95]. The slow step in the overall folding reaction involves the rearrangement of the latter to the fully oxidised native protein containing four disulphide bonds. The Ile56Thr and Asp67His variants were found to fold faster than the wild-type protein by a factor of 2 and 3 respectively, an observation that can be attributed primarily to the reduction in the barriers to conformational rearrangements that results from both the mutations. The efficient folding of these variants despite their enhanced propensities to aggregate when compared to the wild-type protein is consistent with their ability to be secreted in sufficient quantities to give rise to the systemic amyloidoses with which they are associated.