1 Interdisciplinary Nanoscience Center, Science and Technology, Aarhus University2 Department of Biomedicine - Institute of Biophysics, Department of Biomedicine, Health, Aarhus University3 Department of Molecular Biology and Genetics - Department of Molecular Biology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University4 Interdisciplinary Nanoscience Center - INANO-MBG, iNANO-huset, Interdisciplinary Nanoscience Center, Science and Technology, Aarhus University5 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University6 Abbott Products GmbH7 NSM Functional Biomaterials, Roskilde University8 Novozymes A/S9 Interdisciplinary Nanoscience Center - INANO-MBG, iNANO-huset, Interdisciplinary Nanoscience Center, Science and Technology, Aarhus University10 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University
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.