TY - JOUR
T1 - Oxidative folding of human lysozyme
T2 - Effects of the loss of two disulfide bonds and the introduction of a calcium-binding site
AU - Kurokawa, Yousuke
AU - Koganesawa, Nozomi
AU - Kobashigawa, Yoshihiro
AU - Koshiba, Takumi
AU - Demura, Makoto
AU - Nitta, Katsutoshi
PY - 2001/10/16
Y1 - 2001/10/16
N2 - 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)≅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.
AB - 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)≅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.
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U2 - 10.1023/A:1010997500932
DO - 10.1023/A:1010997500932
M3 - Article
C2 - 11594463
AN - SCOPUS:0034816294
SN - 1572-3887
VL - 20
SP - 293
EP - 303
JO - Protein Journal
JF - Protein Journal
IS - 4
ER -