Folding mechanism of mutant human lysozyme C77/95A with increased secretion efficiency in yeast.
A mutant human lysozyme C77/95A, in which Cys77 and Cys95 are replaced with alanine, has been characterized by 8-fold greater secretion in yeast (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967) and almost the same three-dimensional structure as wild-type human lysozyme (Inaka, K., Taniyama, Y., Kikuchi, M., Morikawa, K., and Matsushima, M. (1991) J. Biol. Chem. 266, 12599-12603). To clarify the molecular features of C77/95A and the reason for its increased secretion in yeast, the stabilities of the mutant C77/95A and the wild-type proteins were examined by guanidine hydrochloride denaturation, and the unfolding-refolding kinetics were determined from circular dichroism and fluorescence stopped-flow measurements. Equilibrium experiments showed that the delta G of unfolding of C77/95A in water was 5.8 kcal/mol less stable than that of the wild-type protein at pH 4.0 and 10 degrees C. The unfolding rate of C77/95A was 4 orders of magnitude faster than that of the wild-type protein whereas the two proteins shared similar refolding rates. The slowly refolding phase of the wild-type protein disappeared in C77/95A, indicating that the disulfide bond affects this phase. These observations show that the disulfide bond Cys77-Cys95 contributes to the stabilization of the folded form of human lysozyme by suppressing the unfolding rate and that the increase in the unfolding rate, or the disappearance of the slowly refolding phase in vitro, could correlate with the increase in secretion efficiency in vivo.
Taniyama
Y
Y
Protein Engineering Research Institute, Osaka, Japan.
Ogasahara
K
K
Yutani
K
K
Kikuchi
M
M
D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014961:X-Ray Diffraction
D004220:Disulfides; D009113:Muramidase