title: Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques.
abstract: Protein folding is a thermodynamic process driven by energy gaps between the native and unfolded states. Although a wealth of information is available on the structure of folded species, there is a paucity of data on unfolded species. Here, we analyzed the structural properties of the unfolded state of the starch-binding domain of glucoamylase from Aspergillus niger (SBD) formed in the presence of guanidinium hydrochloride (GuHCl). Although far-UV CD and intrinsic tryptophan fluorescence spectra as well as small angle X-ray scattering suggested that SBD assumes highly unfolded structures in the presence of GuHCl, near-UV circular dichroism of wild-type SBD suggested the presence of residual structures in the unfolded state. Analyses of the unfolded states of tryptophan mutants (W543L, W563A, W590A and W615L) using Similarity Parameter, a modified version of root mean square deviation as a measure of similarity between two spectra, suggested that W543 and W563 have preferences to form native-like residual structures in the GuHCl-unfolded state. In contrast, W615 was entirely unstructured, while W590 tended to form non-native ordered structures in the unfolded state. These data and the amino acid sequence of SBD suggest that local structural propensities in the unfolded state can be determined by the probability of the presence of hydrophobic or charged residues nearby tryptophan residues.
authors.affiliation: Department of Life Science, Faculty of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Ota
authors.affiliation: Department of Bioinformatics, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ikeguchi
authors.affiliation: Department of Life Science, Faculty of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Tanaka
authors.affiliation: Department of Life Science, Faculty of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan; Graduate School of Engineering and Center for Applied Structural Science (CASS), Kobe University, 7-1-48 Minatojima Minami Machi, Chuo-ku, Kobe 650-0047, Japan. Electronic address: daizo@landscape.kobe-u.ac.jp.
authors.forename: Daizo
authors.initials: D
authors.lastname: Hamada
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism / Q000379:methods; D005453:Fluorescence; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D011485:Protein Binding; D011489:Protein Denaturation; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013213:Starch / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D014364:Tryptophan; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; D019791:Guanidine
keywords: Circular dichroism; Fluorescence; Glucoamylase; Protein unfolding; Residual structure; Small angle X-ray scattering