The reversible two-state unfolding of a monocot mannose-binding lectin from garlic bulbs reveals the dominant role of the dimeric interface in its stabilization

Abstract

Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A₂⇔2U). Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding (ΔC_p) and also the temperature dependence of the thermodynamic parameters, namely, ΔG, ΔH, and ΔS. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters ΔH_g, ΔS_g, and ΔC_p as 174.1 kcal mol⁻¹, 0.512 kcal mol⁻¹ K⁻¹, and 3.41 kcal mol⁻¹ K⁻¹, respectively, at T_g=339.4 K. Also, the free energy of unfolding, ΔG_s, at its temperature of maximum stability (T_s=293 K) is 13.13 kcal mol⁻¹. Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined ΔC_p (3.2±0.28 kcal mol⁻¹ K⁻¹) and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small ΔC_p for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein.