Probing the equilibrium denaturation of the serpin α1-antitrypsin with single tryptophan mutants;: Evidence for structure in the urea unfolded state
Probing the equilibrium denaturation of the serpin α1-antitrypsin with single tryptophan mutants;: Evidence for structure in the urea unfolded state
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DOI：
10.1006/jmbi.2001.5104
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发表时间：
2001-11-09
影响因子：
5.6
通讯作者：
Bottomley, SP
中科院分区：
文献类型：
--
作者：
Tew, DJ;Bottomley, SP
The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of alpha (1)-antitrypsin. alpha (1)-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B beta -sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to alpha (1)-antitrypsin. The denaturation of alpha (1-)antitrypsin and its variants was extremely similar when followed by far-UV CID, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A beta -sheet is expanded in the intermediate state whilst no structural change around the B beta -sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed. (C) 2001 Academic Press.
