Oxidative folding of human lysozyme: Effects of the loss of two disulfide bonds and the introduction of a calcium-binding site

Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed t o study the importance of each disulfide bond on oxidative refolding. To av oid destabilization, a calcium-binding site was introduced. Five of the six species of two-disulfide mutants could be obtained with enzymatic activity . Based on the information obtained from refolding and unfolding experiment s, the order of importance in oxidative refolding was found to be as follow s: SS2(Cys30-Cys116) > SS1(Cys6-Cys128) approximate to SS3(Cys65-Cys81) > S S4(Cys77-Cys95). Without SS2, these mutants refolded with low efficiency or did not refold at all. The bond SS2 is located in the interface of B-and D -helices, and a small hydrophobic cluster is formed near SS2. This cluster may play an important role in the folding process and stabilization, and SS 2 may act as a stabilizer through its polypeptide linkage. The bond SS2 is the most important disulfide bond for oxidative folding of lysozymes.