Thermodynamic Effects of Disulfide Bond on Thermal Unfolding of the Starch-Binding Domain of Aspergillus niger Glucoamylase

  title={Thermodynamic Effects of Disulfide Bond on Thermal Unfolding of the Starch-Binding Domain of Aspergillus niger Glucoamylase},
  author={Hayuki Sugimoto and Miho Nakaura and Yoshie Kosuge and Kunio Imai and Hideo Miyake and S Karita and Akiyoshi Tanaka},
  journal={Bioscience, Biotechnology, and Biochemistry},
  pages={1535 - 1541}
The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with β-cyclodextrin at 25 °C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry… Expand
NMR analysis of a kinetically trapped intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase.
Results suggest that, in the intermediate, the aromatic cluster at the surface is structurally less organised, whereas the interior of the protein has relatively rigid, native-like side-chain packing. Expand
Kinetically trapped metastable intermediate of a disulfide‐deficient mutant of the starch‐binding domain of glucoamylase
Results suggest that the intermediate of a thermally unfolded disulfide‐deficient mutant of the starch‐binding domain of glucoamylase could be an off‐pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state. Expand
Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques.
Structural properties of the unfolded state of the starch-binding domain of glucoamylase from Aspergillus niger and the amino acid sequence of SBD suggest that local structural propensities in the unfold state can be determined by the probability of the presence of hydrophobic or charged residues nearby tryptophan residues. Expand
Crystal structure of the starch-binding domain of glucoamylase from Aspergillus niger.
X-ray crystal structural analysis was used to attempt to clarify the conformation of the starch-binding domain of glucoamylase from Aspergillus niger, and it is suggested to be highly flexible. Expand
Novel characteristics of a carbohydrate-binding module 20 from hyperthermophilic bacterium
Tp39E was an independently thermostable CBM and had a considerable effect on APU activity in the hydrolysis of insoluble substrates and bound, to a lesser extent, to soluble and insoluble xylan as well. Expand
The role of thiols and disulfides on protein stability.
The effect of introducing a disulfide bond to improve physical stability of proteins and the mechanisms of degradation of disulfides were discussed and the qualitative/quantitative methods to determine the presence of thiol in the Cys residue and various methods to derivatize thiol group for improving protein stability were illustrated. Expand
The role of thiols and disulfides in protein chemical and physical stability
The use of proteins for therapeutic applications has increased dramatically in the last several decades. There has been a tremendous increase in the number of approved drugs derived from recombinantExpand
The carbohydrate‐binding module family 20 – diversity, structure, and function
The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known starch‐binding domains, and some applications of SBDs as affinity or immobilization tags and, recently, in biofuel and in planta bioengineering are presented. Expand
........................................................................................................................................... ii AcknowledgementsExpand
Fluorescence Spectroscopy in Food Analysis
Food is vital for life, thus it is very important to know how to maintain its quality and thus to detect and analyse all components interfering with food quality. This article describes applicationExpand


Thermal unfolding of the starch binding domain of Aspergillus niger glucoamylase.
A fragment of the starch-binding domain (SBDF) of Aspergillus niger glucoamylase was prepared using recombinant DNA techniques, and its thermal unfolding was investigated by high-sensitivityExpand
Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat.
Two disulfide bridge mutants of the alpha-amylase inhibitor Tendamistat where the large loop or the small loop had been opened by recombinant DNA techniques are investigated, and the stability of the mutated proteins with that of wild-type Tendamists published previously is compared. Expand
Thermodynamic effects of reduction of the active-site disulfide of Escherichia coli thioredoxin explored by differential scanning calorimetry.
The active site of Escherichia coli thioredoxin possesses a disulfide/dithiol in a short loop, oxidation/reduction of which is accompanied by little structural alteration of the protein, and data for the thermal denaturation of the reduced protein are presented. Expand
Enthalpic destabilization of a mutant human lysozyme lacking a disulfide bridge between cysteine-77 and cysteine-95.
Although X-ray crystallography indicated that the mutants preserve the wild-type tertiary structure, removal of the disulfide bridge increased the flexibility of the native state of the mutants, and the effect of cross-linking on the stability of a protein is not solely explained by the entropy change in denaturation. Expand
Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of Interleukin‐4
Analysis of the role of disulfide bridges in IL4 reveals that both mutant proteins have lower conformational stability than the wild‐type protein and plays a critical role in maintaining the thermodynamic stability and core packing of the helix bundle. Expand
Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V
Overall, the results show that an enthalpy‐entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross‐link, and residual structure in theDenatured state. Expand
Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase.
The structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond is studied, and the results suggest that Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Expand
Differential scanning calorimetric studies on the domain structure of Aspergillus glucoamylase.
The thermal unfolding of the starch-binding domain was found to be reversible, but that of the catalytic domain was irreversible. Expand
"Designing out" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor.
Analysis of denaturation transitions for a family of mutants suggests that a previously unrecognized component of disulfide bridge stabilization of proteins is the relatively minor penalty in side chain conformational entropy incurred by cystine residues during folding due to their severely restricted rotation even in the unfolded state. Expand
Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?
The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction. Expand