TAILIEUCHUNG - Báo cáo khoa học: 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. | ễFEBS Journal Role of disulfide bonds in goose-type lysozyme Shunsuke Kawamura1 Mari Ohkuma1 Yuki Chijiiwa1 Daiki Kohno1 Hiroyuki Nakagawa1 Hideki Hirakawa2 Satoru Kuhara2 3 and Takao Torikata1 1 Department of Bioscience Schoolof Agriculture Tokai University Aso Kumamoto Japan 2 Graduate Schoolof Systems Life Sciences Kyushu University Hakozaki Higashi-ku Fukuoka Japan 3 Graduate Schoolof Genetic Resource Technology Kyushu University Hakozaki Higashi-ku Fukuoka Japan Keywords disulfide bonds goose-type lysozyme ostrich site-directed mutagenesis structural stability Correspondence S. Kawamura Department of Bioscience Schoolof Agriculture Tokai University Aso Kumamoto 869-1404 Japan Fax 81 967 67 3960 Tel 81 967 67 3918 E-mail kawamura@ Received 15 November 2007 revised 3 March 2008 accepted 25 March 2008 doi 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

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