Folding of human lysozyme in vivo by the formation of an alternative disulfide bond.
The mutant h-lysozyme, W64CC65A, with Trp64 and Cys65 replaced by Cys and Ala, respectively, was secreted by yeast and purified. Peptide mapping confirmed that W64CC65A contained a nonnative Cys64-Cys81 bond and three native disulfide bonds. The mutant had 2% of the lytic activity of the wild-type lysozyme. The midpoint concentration of the guanidine hydrochloride denaturation curve, the [D]1/2, was 2.7 M for W64CC65A at pH 3.0 and 25 degrees C, whereas the [D]1/2 for the wild-type h-lysozyme was 2.9 M. These results show that the W64CC65A protein is a compactly folded molecule. Our previous results, using the mutant C81A, indicate that Cys81 is not required for correct folding and activity, whereas Cys65 is indispensable (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 65, 7570-7575). Cys64 substituted for Cys65 in W64CC65A, even though the distance between the alpha-carbons at positions 64 and 81 in the wild-type h-lysozyme is not favorable for forming a disulfide bond. Unlike C81A, the mutant W64CC65/81A, which has the additional substitution of Ala for Cys81, did not fold. These results suggest that the absence of both the Cys64-Cys81 bond and the amino acid residue Trp64 caused the misfolding or destabilization of W64CC65/81A in vivo. It is proposed that the formation of the alternative bond, Cys64-Cys81 is important for the folding of W64CC65A in vivo.
Kanaya
E
E
Protein Engineering Research Institute, Osaka, Japan.
Kikuchi
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004271:DNA, Fungal; D004220:Disulfides / Q000737:chemistry*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D004271:DNA, Fungal; D004220:Disulfides; D009113:Muramidase