Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme.
Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.
Taniyama
Y
Y
Protein Engineering Research Institute, Osaka, Japan.
Kuroki
R
R
Omura
F
F
Seko
C
C
Kikuchi
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D014176:Protein Biosynthesis; D015202:Protein Engineering; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014357:Trypsin
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D009113:Muramidase; D014357:Trypsin; D003545:Cysteine