title: Oxidative folding of human lysozyme: effects of the loss of two disulfide bonds and the introduction of a calcium-binding site.
abstract: Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed to study the importance of each disulfide bond on oxidative refolding. To avoid 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 experiments, the order of importance in oxidative refolding was found to be as follows: SS2(Cys30-Cys116) > SS1(Cys6-Cys128) approximately SS3(Cys65-Cys81) > SS4(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 SS2 may act as a stabilizer through its polypeptide linkage. The bond SS2 is the most important disulfide bond for oxidative folding of lysozymes.
authors.affiliation: Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kurokawa
authors.forename: N
authors.initials: N
authors.lastname: Koganesawa
authors.forename: Y
authors.initials: Y
authors.lastname: Kobashigawa
authors.forename: T
authors.initials: T
authors.lastname: Koshiba
authors.forename: M
authors.initials: M
authors.lastname: Demura
authors.forename: K
authors.initials: K
authors.lastname: Niita
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013879:Thioredoxins
chemical_list: D004220:Disulfides; D013879:Thioredoxins; D009113:Muramidase; D002118:Calcium