title: Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments.
abstract: We prepared two dissected fragments of hen lysozyme and examined whether or not these two fragments associated to form a native-like structure. One (Fragment I) is the peptide fragment Asn59-homoserine-105 containing Cys64-Cys80 and Cys76-Cys94. The other (Fragment II) is the peptide fragment Lys1-homoserine-58 connected by two disulfide bridges, Cys6-Cys127 and Cys30-Cys115, to the peptide fragment Asn106-Leu129. It was found that the Fragment I immobilized in the cuvette formed an equimolar complex with Fragment II (K(d) = 3.3x10(-4) M at pH 8 and 25 degrees C) by means of surface plasmon resonance. Moreover, from analyses by circular dichroism spectroscopy and ion-exchange chromatography of the mixture of Fragments I and II at pH 8 under non-reducing conditions, it was suggested that these fragments associated to give the native-like structure. However, the mutant Fragment I in which Cys64-Cys80 and Cys76-Cys94 are lacking owing to the mutation of Cys to Ala, or the mutant fragment in which Trp62 is mutated to Gly, did not form the native-like species with Fragment II, because the mutant Fragment I derived from mutant lysozymes had no local conformation due to mutations. Considering our previous results where the preferential oxidation of two inside disulfide bonds, Cys64-Cys80 and Cys76-Cys94, occurred in the refolding of the fully reduced Fragment I, we suggest that the peptide region corresponding to Fragment I is an initiation site for hen lysozyme folding.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ohkuri
authors.forename: T
authors.initials: T
authors.lastname: Ueda
authors.forename: M
authors.initials: M
authors.lastname: Tsurumaru
authors.forename: T
authors.initials: T
authors.lastname: Imoto
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002645:Chickens; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D004220:Disulfides; D010455:Peptides; D001224:Aspartic Acid; D009113:Muramidase; D008239:Lysine; D003545:Cysteine; D010100:Oxygen; D005998:Glycine