Role of disulfide bonds in goose-type lysozyme.
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 degrees 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.
Kawamura
Shunsuke
S
Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, Japan. kawamura@agri.u-tokai.ac.jp
Ohkuma
Mari
M
Chijiiwa
Yuki
Y
Kohno
Daiki
D
Nakagawa
Hiroyuki
H
Hirakawa
Hideki
H
Kuhara
Satoru
S
Torikata
Takao
T
D000409:Alanine / Q000737:chemistry; D000818:Animals; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D005777:Geese; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D020310:Struthioniformes; D013696:Temperature
D004220:Disulfides; D012694:Serine; D009113:Muramidase; D003545:Cysteine; D000409:Alanine