title: The molten globule of β(2)-microglobulin accumulated at pH 4 and its role in protein folding.
abstract: The acid transition of β(2)-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH4 (25°C). The pH4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH4 MG has been well characterized, although β2m, a β-protein, is structurally very different from α-helical apomyoglobin. NMR pH titration of histidine residues of β2m has also indicated that His84 has an abnormally low pK(a) value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of β2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH4 intermediate. It is proposed that the folding of β2m from its acid-denatured state takes place in the following order: denatured state→MG→burst-phase intermediate→native state.
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: D001059:Apoproteins / Q000737:chemistry; D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009211:Myoglobin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry*
chemical_list: D001059:Apoproteins; D009211:Myoglobin; D010446:Peptide Fragments; C010857:apomyoglobin; D001613:beta 2-Microglobulin; D014364:Tryptophan