# 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

Bachhawat, Kiran and Kapoor, Mili and Dam, Tarun K and Surolia, Avadhesha (2001) 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. In: Biochemistry, 40 (24). pp. 7291-7300.

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## 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 $(A_2 \Longleftrightarrow\hspace{2mm}2U)$. 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 $(\Delta C_p)$ and also the temperature dependence of the thermodynamic parameters, namely, $\Delta G, \Delta H, and \hspace{2mm} \Delta S$. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters $\Delta H_g$, $\Delta S_g,$ and $\Delta C_p$ as $174.1 \hspace{2mm} kcal\hspace{1mm} mol^{-1}$, $0.512 \hspace{2mm}kcal \hspace{1mm}mol^{-1} K^{-1}$, and $3.41 \hspace{2mm}kcal \hspace{1mm} mol^{-1} K^{-1}$, respectively, at $T_g$ = 339.4 K. Also, the free energy of unfolding, $G_s,$ at its temperature of maximum stability $(T_s = 293 K)$ is $13.13 \hspace{2mm} kcal\hspace{1mm} mol^{-1}$. Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined $C_p (3.2 \pm 0.28 \hspace{2mm}kcal \hspace{1mm}mol^{-1} K^{-1})$ 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 $C_p$ for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein.

Item Type: Journal Article Copyright of this article belongs to American Chemical Society(ACS). Biochemistry;Molecular Biophysics;Methods and Techniques;Pharmacognosy;Pharmacology Division of Biological Sciences > Molecular Biophysics Unit 03 Oct 2007 19 Sep 2010 04:39 http://eprints.iisc.ernet.in/id/eprint/12027