Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques

July 28, 2017

Title

Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques

Author

Chiaki Ota, Masamichi Ikeguchi, Akiyoshi Tanaka, Daizo Hamada

Year

2016

Journal

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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.

Instrument

J-720

Keywords

Circular dichroism, Secondary structure, Tertiary structure, Protein folding, Protein denaturation, Thermodynamics, Biochemistry