title: Characterization of a human alpha1-antitrypsin variant that is as stable as ovalbumin.
abstract: The metastability of inhibitory serpins (serine proteinase inhibitors) is thought to play a key role in the facile conformational switch and the insertion of the reactive center loop into the central beta-sheet, A-sheet, during the formation of a stable complex between a serpin and its target proteinase. We have examined the folding and inhibitory activity of a very stable variant of human alpha1-antitrypsin, a prototype inhibitory serpin. A combination of seven stabilizing single amino acid substitutions of alpha1-antitrypsin, designated Multi-7, increased the midpoint of the unfolding transition to almost that of ovalbumin, a non-inhibitory but more stable serpin. Compared with the wild-type alpha1-antitrypsin, Multi-7 retarded the opening of A-sheet significantly, as revealed by the retarded unfolding and latency conversion of the native state. Surprisingly, Multi-7 alpha1-antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of inhibition as the wild type, indicating that enhanced A-sheet closure conferred by Multi-7 does not affect the complex formation. It may be that the stability increase of Multi-7 alpha1-antitrypsin is not sufficient to influence the rate of loop insertion during the complex formation.
authors.affiliation: Division of Protein Engineering, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305-600, Korea.
authors.forename: K N
authors.initials: KN
authors.lastname: Lee
authors.forename: H
authors.initials: H
authors.lastname: Im
authors.forename: S W
authors.initials: SW
authors.lastname: Kang
authors.forename: M H
authors.initials: MH
authors.lastname: Yu
mesh_terms: D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010047:Ovalbumin / Q000737:chemistry; D010196:Pancreatic Elastase / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
chemical_list: D000515:alpha 1-Antitrypsin; D010047:Ovalbumin; D010196:Pancreatic Elastase