Nonlocal interactions are responsible for tertiary structure formation in staphylococcal nuclease.
Rapid molecular collapse mediated by nonlocal interactions is believed to be a crucial event for protein folding. To investigate the role of nonlocal interactions in tertiary structure formation, we performed a nonlocal interaction substitution mutation analysis on staphylococcal nuclease (SNase). Y54 and I139 of wild-type (WT) SNase and Delta140-149 were substituted by cysteine to form intramolecular disulfide bonds, respectively called WT-SS and Delta140-149-SS. Under physiological conditions, the reduced form of Delta140-149-SS appears to assume a denatured structure; in contrast, the oxidized form of Delta140-149-SS forms a native-like structure. From this result, we conclude that the C-terminal region participates in a nonlocal interaction that is indispensable for the native structure. Although the oxidized form of WT-SS assumes a more compact denatured structure under acidic conditions than the WT, the kinetic measurements reveal that the refolding reactions of both the reduced and oxidized forms of WT-SS are similar to those of the WT, suggesting that an intact nonlocal interaction is established within the dead time (22 ms). On the basis of these results, we propose that the native nonlocal contact established at the early stage of the folding process facilitates further secondary structure formation.
Graduate School of Materials Science, Nara Institute of Science and Technology, Nara, Japan.
Shingo
S
Kato
Hironari
H
Kamikubo
Satoshi
S
Hirano
Yoichi
Y
Yamazaki
Mikio
M
Kataoka
D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012996:Solutions; D013696:Temperature
D004220:Disulfides; D012996:Solutions; D008836:Micrococcal Nuclease