pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus

Huabing Wang, Kell Kleiner Andersen, Pankaj Sehgal, J Hagedorn, Peter Westh, Kim Borch, Daniel Otzen

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    Resumé

    Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4–6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately −12 to approximately −5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.
    OriginalsprogEngelsk
    TidsskriftBiochemistry
    Vol/bind52
    Udgave nummer1
    Sider (fra-til)264-276
    ISSN0006-2960
    DOI
    StatusUdgivet - 8 jan. 2013

    Citer dette

    Wang, H., Andersen, K. K., Sehgal, P., Hagedorn, J., Westh, P., Borch, K., & Otzen, D. (2013). pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus. Biochemistry, 52(1), 264-276. https://doi.org/10.1021/bi301258e
    Wang, Huabing ; Andersen, Kell Kleiner ; Sehgal, Pankaj ; Hagedorn, J ; Westh, Peter ; Borch, Kim ; Otzen, Daniel. / pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus. I: Biochemistry. 2013 ; Bind 52, Nr. 1. s. 264-276.
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    abstract = "Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4–6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately −12 to approximately −5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.",
    author = "Huabing Wang and Andersen, {Kell Kleiner} and Pankaj Sehgal and J Hagedorn and Peter Westh and Kim Borch and Daniel Otzen",
    year = "2013",
    month = "1",
    day = "8",
    doi = "10.1021/bi301258e",
    language = "English",
    volume = "52",
    pages = "264--276",
    journal = "Biochemistry",
    issn = "0006-2960",
    publisher = "American Chemical Society",
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    Wang, H, Andersen, KK, Sehgal, P, Hagedorn, J, Westh, P, Borch, K & Otzen, D 2013, 'pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus', Biochemistry, bind 52, nr. 1, s. 264-276. https://doi.org/10.1021/bi301258e

    pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus. / Wang, Huabing; Andersen, Kell Kleiner; Sehgal, Pankaj; Hagedorn, J; Westh, Peter; Borch, Kim; Otzen, Daniel.

    I: Biochemistry, Bind 52, Nr. 1, 08.01.2013, s. 264-276.

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    TY - JOUR

    T1 - pH Regulation of the Kinetic Stability of the Lipase from Thermomyces lanuginosus

    AU - Wang, Huabing

    AU - Andersen, Kell Kleiner

    AU - Sehgal, Pankaj

    AU - Hagedorn, J

    AU - Westh, Peter

    AU - Borch, Kim

    AU - Otzen, Daniel

    PY - 2013/1/8

    Y1 - 2013/1/8

    N2 - Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4–6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately −12 to approximately −5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.

    AB - Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4–6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately −12 to approximately −5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.

    U2 - 10.1021/bi301258e

    DO - 10.1021/bi301258e

    M3 - Journal article

    VL - 52

    SP - 264

    EP - 276

    JO - Biochemistry

    JF - Biochemistry

    SN - 0006-2960

    IS - 1

    ER -