Probing the equilibrium denaturation of the serpin α1-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state
Identifieur interne : 003520 ( Main/Exploration ); précédent : 003519; suivant : 003521Probing the equilibrium denaturation of the serpin α1-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state
Auteurs : Deborah J. Tew ; Stephen P. Bottomley [Australie]Source :
- Journal of Molecular Biology [ 0022-2836 ] ; 2001.
English descriptors
- KwdEn :
- Teeft :
- Academic press, Antitrypsin, Biochemistry, Biol, Carrell, Chem, Conformation, Conformational, Conformational change, Conformational changes, Conformational disease, Denaturant, Denaturation, Denatured state, Different denaturants, Early events, Emission intensity, Emission slit widths, Excitation, Excitation wavelength, Fluorescence emission spectra, Free energy change, Free energy changes, Gdnscn, Genetic variants, Guanidine thiocyanate, Hydrophobic residues, Individual curves, Inhibitory mechanism, Inhibitory properties, Intermediate conformation, Intermediate state, Linear extrapolation method, Liver disease, Lmax, Lmax value, Lomas, Metastable state, Midpoint, Mutation, Native state, Natl acad, Nature struct, Other studies, Pathway, Phenylalanine substitutions, Proteinase, Reactive, Reactive center loop, Residual structure, Second transition, Secondary structure, Serine proteinase inhibitor, Serpin, Serpin superfamily, Serpins, Single tryptophan mutants, Single tryptophan variants, Structural changes, Superfamily, Thermal stability, Tryptophan, Tryptophan residues, Uorescence, Uorescence emission intensity, Uorescence emission spectra, Uorescence spectroscopy, Urea, Urea concentration, Variant.
Abstract
Abstract: 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 α1-antitrypsin. α1-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B β-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 α1-antitrypsin. The denaturation of α1-antitrypsin and its variants was extremely similar when followed by far-UV CD, 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 β-sheet is expanded in the intermediate state whilst no structural change around the B β-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.
Url:
DOI: 10.1006/jmbi.2001.5104
Affiliations:
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Probing the equilibrium denaturation of the serpin α1-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state</title><author><name sortKey="Tew, Deborah J" sort="Tew, Deborah J" uniqKey="Tew D" first="Deborah J" last="Tew">Deborah J. Tew</name></author><author><name sortKey="Bottomley, Stephen P" sort="Bottomley, Stephen P" uniqKey="Bottomley S" first="Stephen P" last="Bottomley">Stephen P. 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In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of α1-antitrypsin. α1-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B β-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 α1-antitrypsin. The denaturation of α1-antitrypsin and its variants was extremely similar when followed by far-UV CD, 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 β-sheet is expanded in the intermediate state whilst no structural change around the B β-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.</div></front></TEI><affiliations><list><country><li>Australie</li></country></list><tree><noCountry><name sortKey="Tew, Deborah J" sort="Tew, Deborah J" uniqKey="Tew D" first="Deborah J" last="Tew">Deborah J. Tew</name></noCountry><country name="Australie"><noRegion><name sortKey="Bottomley, Stephen P" sort="Bottomley, Stephen P" uniqKey="Bottomley S" first="Stephen P" last="Bottomley">Stephen P. Bottomley</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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