title: Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate.
abstract: We investigated the role of a partially folded intermediate that transiently accumulates during lysozyme folding. Previous studies had shown that the partially folded intermediate is located on a slow-folding pathway and that an additional fast direct pathway from the unfolded state to the native state exists. Kinetic double-jump experiments showed that the two folding pathways are not caused by slow equilibration reactions in the unfolded state. Rather, kinetic partitioning occurs very early in lysozyme refolding, giving the molecules the chance to enter the direct pathway or a slow-folding channel. Fitting the guanidinium chloride dependencies of the refolding and unfolding reactions to analytical solutions for different folding scenarios enables us to propose a triangular mechanism as the minimal model for lysozyme folding explaining all observed kinetic reactions: [diagram in text]. All microscopic rate constants and their guanidinium chloride dependencies could be obtained from the experimental data. The results suggest that population of the intermediate during refolding increases the free energy of activation of the folding process. This effect is due to the increased stability of the intermediate state compared to the unfolded state leading to an increase in the free energy of activation (deltaG0) compared to folding in the absence of populated intermediate states. The absolute energy of the transition state is identical on both pathways. The results imply that pre-formed secondary structure in the folding intermediate obstructs formation of the transition state of folding but does not change the nature of the rate-limiting step in the folding process.
authors.affiliation: Department of Biophysical Chemistry, Biozentrum der Universität Basel,Switzerland.
authors.forename: G
authors.initials: G
authors.lastname: Wildegger
authors.forename: T
authors.initials: T
authors.lastname: Kiefhaber
mesh_terms: D000818:Animals; D002645:Chickens; D005260:Female; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol / Q000494:pharmacology
chemical_list: D006146:Guanidines; D014270:Trifluoroethanol; D009113:Muramidase; D019791:Guanidine