title: Native-state heterogeneity of β(2)-microglobulin as revealed by kinetic folding and real-time NMR experiments.
abstract: The kinetic folding of β(2)-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH7.5 to pH2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH2.0 to pH7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5-6 s(-1), while the molecule with the Pro32 trans isomer refolds more slowly (pH7.5 and 25°C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001-0.002 s(-1)) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7-9% under a more physiological condition (pH7.5 and 37°C).
authors.affiliation: Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Mukaiyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
mesh_terms: D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics; D001613:beta 2-Microglobulin / Q000737:chemistry*
chemical_list: D001613:beta 2-Microglobulin