Formation of alpha-helix 88-98 is essential in the establishment of higher-order structure from reduced lysozyme.
Lys96 and Lys97 in lysozyme are located at the C terminus of alpha-helix 88-98. The positive charges of these residues are supposed to stabilize the helical structure, and these residues are conserved as the basic amino acids among c-type lysozymes. The renaturation rate of reduced mutant lysozyme, where both Lys96 and Lys97 were mutated together to Ala, was slower than that of native lysozyme at pH 8.0 and 37 degrees C by SH-SS interchange reactions. In order to investigate the reason, the peptide fragment 36-105 (where we can obtain information of the interaction between helix 88-98 and Trp62 and Trp63 residues) was prepared. CD spectra were compared between peptide fragment 36-105 and its acetylated form, where the positive charges of Lys96 and Lys97 were eliminated, and it was elucidated that the displacement of positive charges at the C terminus of the helix caused the shift of the advantageous structure of the fragment from alpha-helix to coil. Moreover, we obtained evidence that there was interaction of the helix with Trp62 and/or Trp63, which maintained a thermodynamically stable higher-order structure. Therefore, these results suggest that the formation of alpha-helical structure in 88-98 is a significant factor in the establishment of native structure from reduced lysozyme.
Ueda
T
T
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Nakashima
A
A
Hashimoto
Y
Y
Miki
T
T
Yamada
H
H
Imoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004247:DNA; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D010446:Peptide Fragments; D004247:DNA; D009113:Muramidase; D008239:Lysine