Baek, Je-Hyun Yang, Won Suk Lee, Cheolju Yu, Myeong-Hee 2024-01-20T21:32:48Z 2024-01-20T21:32:48Z 2021-09-01 2009-05 1535-9476 https://pubs.kist.re.kr/handle/201004/132549 The native state of alpha(1)-antitrypsin (alpha(1)AT), 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 alpha(1)AT 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 beta-trypsin. Comparing the deuterium content at the corresponding regions of these three samples, we probed the unfolding of alpha(1)AT during complex formation. A substantial portion of the alpha(1)AT 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 beta-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. Molecular & Cellular Proteomics 8: 1072-1081, 2009. English AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC PLASMINOGEN-ACTIVATOR INHIBITOR-1 SERPIN-PROTEINASE COMPLEX STRUCTURAL BASIS HYDROGEN/DEUTERIUM EXCHANGE CONFORMATIONAL STABILITY SUICIDE SUBSTRATE NATIVE STRAIN REACTIVE LOOP KINETIC TRAP MECHANISM Functional Unfolding of alpha(1)-Antitrypsin Probed by Hydrogen-Deuterium Exchange Coupled with Mass Spectrometry Article 10.1074/mcp.M800365-MCP200 1 MOLECULAR & CELLULAR PROTEOMICS, v.8, no.5, pp.1072 - 1081 MOLECULAR & CELLULAR PROTEOMICS 8 5 1072 1081 scie scopus 000266116900017 2-s2.0-67449100204 Biochemical Research Methods Biochemistry & Molecular Biology Article PLASMINOGEN-ACTIVATOR INHIBITOR-1 SERPIN-PROTEINASE COMPLEX STRUCTURAL BASIS HYDROGEN/DEUTERIUM EXCHANGE CONFORMATIONAL STABILITY SUICIDE SUBSTRATE NATIVE STRAIN REACTIVE LOOP KINETIC TRAP MECHANISM