title: Spontaneous asparaginyl deamidation of canine milk lysozyme under mild conditions.
abstract: Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.
authors.affiliation: Graduate School of Science, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nonaka
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.forename: Daisuke
authors.initials: D
authors.lastname: Akieda
authors.forename: Masanori
authors.initials: M
authors.lastname: Yasui
authors.forename: Masahiro
authors.initials: M
authors.lastname: Watanabe
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Watanabe
authors.forename: Isao
authors.initials: I
authors.lastname: Tanaka
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Kamiya
authors.forename: Mineyuki
authors.initials: M
authors.lastname: Mizuguchi
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kawano
mesh_terms: D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004285:Dogs; D004795:Enzyme Stability; D006207:Half-Life; D008892:Milk / Q000201:enzymology*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D000577:Amides; D050505:Mutant Proteins; D001216:Asparagine; D009113:Muramidase