Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments.
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.
Ohkuri
T
T
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Ueda
T
T
Tsurumaru
M
M
Imoto
T
T
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
D004220:Disulfides; D010455:Peptides; D001224:Aspartic Acid; D009113:Muramidase; D008239:Lysine; D003545:Cysteine; D010100:Oxygen; D005998:Glycine