Thermal, chemical and pH induced denaturation of a multimeric β-galactosidase reveals multiple unfolding pathways

Abstract

Background: In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH.

Methodology/principal findings: CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50 °C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG(0)) and unfolding constant (K(obs)) were also calculated for chemically denatured CpGAL.

Significance: The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well.

Figure 1. Native spectra for CpGAL.

(A) Represents Far UV CD spectra, whereas (B) represents fluorescence emission spectra at different excitation wavelengths.

Figure 2. Thermal denaturation.

Ellipticity (A & B) and emission (C & D) measurement of CpGAL as a function of temperature. Emission shows monotonous decrease in the intensity of λ_max with increasing temperature. (E) Represents residual enzymatic activity as a function of heat denaturation for 5 min, at respective temperatures.

Figure 3. pH induced denaturation.

Ellipticity (A), emission (B) and enzymatic activity (C) measurement of CpGAL as a function of pH. The enzymatic activity was estimated at respective pH of incubation and at pH 2.8 (according to the standard activity assay protocol). (D) Represents fluorescence intensity of ANS binding. (E) Represents size exclusion chromatography (SEC) profile of CpGAL at pH 2.0 and pH 4.0.

Figure 4. Chemical induced conformational changes.

(A) & (B) represents GdHCl and urea mediated transitions of protein unfolding, respectively. (C) Represents free energy of unfolding, whereas (D) represents unfolding kinetics rate constants (Half Chevron plot) against GdHCl. Intercepts at y axis gives ΔG₀ and log K_obs, respectively.

Figure 5. Proposed denaturation scheme for CpGAL.

The enzyme uses different pathways of unfolding for different denaturants.