Comprehensive secondary-structure analysis of disulfide variants of lysozyme by synchrotron-radiation vacuum-ultraviolet circular dichroism.
To elucidate the effects of specific disulfide bridges (Cys6-Cys127, Cys30-Cys115, Cys64-Cys80, and Cys76-Cys94) on the secondary structure of hen lysozyme, the vacuum-ultraviolet circular dichroism (VUVCD) spectra of 13 species of disulfide-deficient variants in which Cys residues were replaced with Ala or Ser residues were measured down to 170 nm at pH 2.9 and 25 degrees C using a synchrotron-radiation VUVCD spectrophotometer. Each variant exhibited a VUVCD spectrum characteristic of a considerable amount of residual secondary structures depending on the positions and numbers of deleted disulfide bridges. The contents of alpha-helices, beta-strands, turns, and unordered structures were estimated with the SELCON3 program using the VUVCD spectra and PDB data of 31 reference proteins. The numbers of alpha-helix and beta-strand segments were also estimated from the VUVCD data. In general, the secondary structures were more effectively stabilized through entropic forces as the number of disulfide bridges increased and as they were formed over larger distances in the primary structure. The structures of three-disulfide variants were similar to that of the wild type, but other variants exhibited diminished alpha-helices with a border between the ordered and disordered structures around the two-disulfide variants. The sequences of the secondary structures were predicted for all the variants by combining VUVCD data with a neural-network method. These results revealed the characteristic role of each disulfide bridge in the formation of secondary structures.
Hiroshima Synchrotron Radiation Center, Hiroshima University, Higashi-Hiroshima 739-0046, Japan.
Koichi
K
Matsuo
Hidenori
H
Watanabe
Shin-ichi
S
Tate
Hideki
H
Tachibana
Kunihiko
K
Gekko
D000818:Animals; D002942:Circular Dichroism / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D009113:Muramidase / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017356:Synchrotrons
D004220:Disulfides; D009113:Muramidase