Analysis of the stabilization of hen lysozyme by helix macrodipole and charged side chain interaction

In the N-terminal region of the a-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Aspl8/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asnl8/Asn27 (18N/27N), Asnl8/Asp27 (18N/27D), and Aspl8/ Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Aspl8 and the amide group at the Nl position in the a-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Aspl8 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein. Key words: hen lysozyme, NMB, site-directed mutagenesis, stability, X-ray crystallogra-phy. A degree of instability of proteins is important for physio