title: Kinetically trapped metastable intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase.
abstract: Refolding of a thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and (1)H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half-lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with beta-cyclodextrin as the native state, suggesting that the intermediate is highly-ordered and native-like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far-UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off-pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.
authors.affiliation: Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan.
authors.forename: Hayuki
authors.initials: H
authors.lastname: Sugimoto
authors.forename: Miho
authors.initials: M
authors.lastname: Nakaura
authors.forename: Shigenori
authors.initials: S
authors.lastname: Nishimura
authors.forename: Shuichi
authors.initials: S
authors.lastname: Karita
authors.forename: Hideo
authors.initials: H
authors.lastname: Miyake
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Tanaka
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000502:physiology; D013057:Spectrum Analysis; D047392:beta-Cyclodextrins / Q000378:metabolism*
chemical_list: D004220:Disulfides; D047392:beta-Cyclodextrins; D005087:Glucan 1,4-alpha-Glucosidase