Premium
Role of disulfide bonds in goose‐type lysozyme
Author(s) -
Kawamura Shunsuke,
Ohkuma Mari,
Chijiiwa Yuki,
Kohno Daiki,
Nakagawa Hiroyuki,
Hirakawa Hideki,
Kuhara Satoru,
Torikata Takao
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06422.x
Subject(s) - lysozyme , mutant , chemistry , guanidine , disulfide bond , protein folding , protein disulfide isomerase , folding (dsp implementation) , wild type , stereochemistry , cysteine , protein secondary structure , crystallography , biochemistry , enzyme , electrical engineering , gene , engineering
The role of the two disulfide bonds (Cys4–Cys60 and Cys18–Cys29) in the activity and stability of goose‐type (G‐type) lysozyme was investigated using ostrich egg‐white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild‐type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild‐type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G‐type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild‐type was decreased to a level nearly equivalent to that of a Cys‐free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys‐free mutant was downshifted by about 20 °C as compared with that of the wild‐type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G‐type lysozyme.