Native-like tertiary structure formation in the alpha-domain of a hen lysozyme two-disulfide variant.
Structure formation in two species of the two-disulfide variant of hen lysozyme was investigated by means of CD spectroscopy, disulfide exchange measurement, and 1H-NMR spectroscopy. One species, 2SS [6-127, 30-115], which contained the two disulfide bonds found in the alpha-domain of authentic lysozyme, had amounts of secondary and tertiary structures, and bacteriolytic activity comparable to those of authentic lysozyme, and showed a cooperative thermal unfolding. By contrast, the other species, 2SS [64-80, 76-94], which contained the beta-domain disulfide bond as well as the inter-domain one, had a limited amount of secondary structure and little tertiary structure. Disulfide-exchange did not occur for 2SS [6-127, 30-115], whereas it occurred for 2SS [64-80, 76-94], indicating that the protein main-chain fold coupled with the formation of two disulfide bonds is relatively stable for the former variant, while unstable for the latter. 1H-NMR spectra of 2SS [6-127, 30-115] showed that native-like local environment is present within the region that corresponds to the alpha-domain, while it is absent within the region that corresponds to the beta or inter-domain. These results indicate that the alpha-domain of hen lysozyme can be an independent folding domain at equilibrium. Although the bipartite nature in the structure formation of hen lysozyme is similar to that reported for alpha-lactalbumin, differences exist between the disulfide-intermediates of the two proteins in terms of the structural domain that accomplishes tertiary structure.
Tachibana
H
H
Department of Biology, Kobe University, Japan. tachiban@biol.sci.kobe-u.ac.jp
Oka
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T
Akasaka
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K
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D002645:Chickens / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D005260:Female; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D016982:Micrococcus luteus / Q000378:metabolism; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D004220:Disulfides; D007768:Lactalbumin; D009113:Muramidase; D003545:Cysteine