Analysis of the early stage of the folding process of reduced lysozyme using all lysozyme variants containing a pair of cysteines.
Twenty-eight hen lysozyme variants that contained a pair of cysteines were constructed to examine the formation of the individual native and nonnative disulfide bonds. We analyzed the extent of the formation of a disulfide bond in each lysozyme variant using a redox buffer (pH 8) containing 1.0 mM reduced and 0.1 mM oxidized glutathione in the absence or presence of 6 M guanidine hydrochloride. In the presence of 6 M guanidine hydrochloride, the extent of the formation of the disulfide bond in each lysozyme variant was proportional to the distance between cysteine residues, indicating that reduced hen lysozyme under a highly denaturing condition adopted a randomly coiled structure. In aqueous solution, the formations of all disulfide bonds occurred much more easily than under a denatured condition. This finding indicated that reduced lysozyme had a somewhat compact structure. Moreover, the scattering data for the extents of the formation of the disulfide bonds among all lysozyme variants were observed. These results suggested that the nonrandom folding occurred in the early stage of the folding of reduced lysozyme, which should provide new insight into the early-stage events in the folding process of reduced lysozyme.
Shioi
Seijiro
S
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Imoto
Taiji
T
Ueda
Tadashi
T
D000818:Animals; D002645:Chickens; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D019277:Entropy; D014644:Genetic Variation; D019791:Guanidine / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012996:Solutions; D014867:Water
D004220:Disulfides; D011994:Recombinant Proteins; D012996:Solutions; D014867:Water; C421864:hen egg lysozyme; D009113:Muramidase; D019791:Guanidine; D003545:Cysteine