title: The equilibrium unfolding intermediate observed at pH 4 and its relationship with the kinetic folding intermediates in green fluorescent protein.
abstract: Folding mechanisms of a variant of green fluorescent protein (F99S/M153T/V163A) were investigated by a wide variety of spectroscopic techniques. Equilibrium measurements on acid-induced denaturation of the protein monitored by chromophore and tryptophan fluorescence and small-angle X-ray scattering revealed that this protein accumulates at least two equilibrium intermediates, a native-like intermediate and an unfolding intermediate, the latter of which exhibits the characteristics of the molten globule state under moderately denaturing conditions at pH 4. To elucidate the role of the equilibrium unfolding intermediate in folding, a series of kinetic refolding experiments with various combinations of initial and final pH values, including pH 7.5 (the native condition), pH 4.0 (the moderately denaturing condition where the unfolding intermediate is accumulated), and pH 2.0 (the acid-denaturing condition) were carried out by monitoring chromophore and tryptophan fluorescence. Kinetic on-pathway intermediates were accumulated during the folding on the refolding reaction from pH 2.0 to 7.5. However, the signal change corresponding to the conversion from the acid-denatured to the kinetic intermediate states was significantly reduced on the refolding reaction from pH 4.0 to pH 7.5, whereas only the signal change corresponding to the above conversion was observed on the refolding reaction from pH 2.0 to pH 4.0. These results indicate that the equilibrium unfolding intermediate is composed of an ensemble of the folding intermediate species accumulated during the folding reaction, and thus support a hierarchical model of protein folding.
authors.affiliation: Department of Physics, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors.forename: Sawako
authors.initials: S
authors.lastname: Enoki
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.forename: Tomonao
authors.initials: T
authors.lastname: Inobe
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Takahashi
authors.forename: Kiyoto
authors.initials: K
authors.lastname: Kamagata
authors.forename: Tomotaka
authors.initials: T
authors.lastname: Oroguchi
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Nakatani
authors.forename: Katsuaki
authors.initials: K
authors.lastname: Tomoyori
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
mesh_terms: D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D014364:Tryptophan / Q000737:chemistry; D014965:X-Rays
chemical_list: D050505:Mutant Proteins; D049452:Green Fluorescent Proteins; D014364:Tryptophan