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Probing the local conformational change of α1-antitrypsin

Authors

  • Je-Hyun Baek,

    1. Functional Proteomics Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Korea
    2. Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul 136-701, Korea
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  • Hana Im,

    1. Department of Molecular Biology, Sejong University, Gwangjin-gu, Seoul 143-747, Korea
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  • Un-Beom Kang,

    1. Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul 136-701, Korea
    2. Life Sciences Division, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Korea
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  • Ki Moon Seong,

    1. Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul 136-701, Korea
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  • Cheolju Lee,

    1. Life Sciences Division, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Korea
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  • Joon Kim,

    Corresponding author
    1. Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seongbuk-gu, Seoul 136-701, Korea
    • School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Korea; fax: +82-2-927-9028.
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  • Myeong-Hee Yu

    Corresponding author
    1. Functional Proteomics Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Korea
    • Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Korea; fax: +82-2-958-6919
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Abstract

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of β-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in α1-antitrypsin, which would lock the displacement of thFs3A from β-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop–sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop–sheet polymerization significantly. This successful protein engineering of α1-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into β-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.

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