title: pH-dependent unfolding of aspergillopepsin II studied by small-angle X-ray scattering.
abstract: Aspergillopepsin II (EC 3.4.23.6) secreted from the fungus Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase. It consists of two polypeptide chains (i.e., a heavy chain and a light chain), which are bound noncovalently to each other. The pH titration analysis using small-angle X-ray scattering (SAXS) as well as circular dichroism (CD) and gel filtration indicated that the enzyme was unfolded around a neutral pH with concomitant dissociation of the two chains. Detailed analyses showed that the midpoint pH values for the unfolding are not coincident with one another (pH 6.1 in circular dichroism and gel filtration, pH 6.4 in zero-angle intensity of SAXS, pH 6.8 in radius of gyration). The difference between these values suggested the existence of an intermediate state during the unfolding. Further analyses of the SAXS data showed that the heavy chain just after the dissociation still kept molecular compactness and that it gradually increased its dimensions as the pH was further raised. Noncoincidence of the two phenomena (i.e., chain dissociation and swelling) led to elucidation of a novel intermediate state during unfolding, which was confirmed by the subsequent singular value decomposition (SVD) analysis.
authors.affiliation: School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kojima
authors.forename: M
authors.initials: M
authors.lastname: Tanokura
authors.forename: M
authors.initials: M
authors.lastname: Maeda
authors.forename: K
authors.initials: K
authors.lastname: Kimura
authors.forename: Y
authors.initials: Y
authors.lastname: Amemiya
authors.forename: H
authors.initials: H
authors.lastname: Kihara
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013816:Thermodynamics; D014965:X-Rays
chemical_list: D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II