title: Relationship between the stability of hen egg-white lysozymes mutated at sites designed to interact with alpha-helix dipoles and their secretion amounts in yeast.
abstract: The positively charged lysine at the C-terminals of three long alpha-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change Delta G of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long alpha-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and Delta G of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long alpha-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.
authors: Department of Biological Chemistry, Yamaguchi University, Yamaguchi, Japan.
authors: Akihito
authors: A
authors: Harada
authors: Hiroshi
authors: H
authors: Yagi
authors: Akira
authors: A
authors: Saito
authors: Hiroyuki
authors: H
authors: Azakami
authors: Akio
authors: A
authors: Kato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010843:Pichia / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: C421864:hen egg lysozyme; D009113:Muramidase