Functional unfolding of α1-antitrypsin probed by hydrogen-deuterium exchange coupled with mass spectrometry

Abstract

The native state of α1-antitrypsin (α1AT) , a member of the serine protease inhibitor (serpin) family, is considered a kinetically trapped folding intermediate that converts to a more stable form upon complex formation with a target protease. Although previous structural and mutational studies of α1AT revealed the structural basis of the native strain and the kinetic trap, the mechanism of how the native molecule overcomes the kinetic barrier to reach the final stable conformation during complex formation remains unknown. We hypothesized that during complex formation, a substantial portion of the molecule undergoes unfolding, which we dubbed functional unfolding. Hydrogen-deuterium exchange coupled with ESI-MS was used to analyze this serpin in three forms: native, complexing, and complexed with bovine β-trypsin. Comparing the deuterium content at the corresponding regions of these three samples, we probed the unfolding of α 1AT during complex formation. A substantial portion of the α1AT molecule unfolded transiently during complex formation, including not only the regions expected from previous structural studies, such as the reactive site loop, helix F, and the following loop, but also regions not predicted previously, such as helix A, strand 6 of β-sheet B, and the N terminus. Such unfolding of the native interactions may elevate the free energy level of the kinetically trapped native serpin sufficiently to cross the transition state during complex formation. In the current study, we provide evidence that protein unfolding has to accompany functional execution of the protein molecule. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.