Fig 1.
Homology model of wheat α-amylase, with aromatic residues highlighted in the following colors: tryptophan—green; tyrosine—cyan; phenylalanine—light blue.
The enzyme displayed 13 tryptophan, 15 tyrosine and 16 phenylalanine side chains.
Fig 2.
Spectroscopic properties of α-amylase.
(A) Native far-UV CD spectrum and (B) Fluorescence emission spectra at different excitation wavelengths of α-amylase reveal similar emission maximum (tryptophan, 340 nm) with higher energy emission at lower excitation wavelengths (as both tyrosine and tryptophan are excited) whereas lower emission at higher wavelength (only tryptophan being excited).
Fig 3.
Thermal denaturation profiles of α-amylase (A) Residual enzymatic activity as a function of heat denaturation was estimated both at respective temperature of incubation (open circle) and at 68°C followed by incubation for 5 min at various temperatures (closed circle) (B) Far-UV CD spectra at various temperatures (C) Changes in ellipticity at 222 nm with temperature (D) Temperature-dependent fluorescence spectra of α-amylase.
Fig 4.
pH induced denaturation profiles of α-amylase (A) α-Amylase activity as a function of pH.
The enzymatic activity was estimated both at respective pH of incubation (red) and at pH 5.0 followed by incubation at various pH (black) (B) Changes in ellipticity at 222 nm with pH (C) pH dependent fluorescence spectra of α-amylase (D) Fluorescence intensity of ANS binding to α-amylase at different pH.
Fig 5.
Chemical induced conformational changes of α-amylase (A) and (B) represents GdHCl and urea mediated transitions of protein unfolding, respectively (C) Unfolding kinetics rate constants (Half-Chevron plot) against GdHCl (D) Free energy of unfolding.
Intercepts at y axis returns log Kobs and ΔG0, respectively.
Table 1.
Thermodynamic and kinetic parameters for unfolded α-amylase.
Fig 6.
Schematic representation of possible unfolding pathways undertaken by α-amylase during treatment with different denaturants.