Conformational Stability of Legume Lectins Reflect Their Different Modes of Quaternary Association:  Solvent Denaturation Studies on Concanavalin A and Winged Bean Acidic Agglutinin

Abstract

Thermodynamic parameters associated with the unfolding of the legume lectin, WBA II, were determined by isothermal denaturation. The analysis of isothermal denaturation data provided values for conformational stability and heat capacity for WBA II unfolding. To explore the role of intersubunit contact in stability, we carried out similar studies under identical conditions on Concanavalin A, a legume lectin of nearly similar size, buried hydrophobic surface area and tertiary structure to that of WBA II but with a different oligomerization pattern. Both proteins showed a reversible two-state unfolding with guanidine hydrochloride. As expected, the change in heat capacity upon unfolding was similar for both proteins at 3.5 and 3.7 kcal mol(-1) K(-1) for Concanavalin A and WBA II, respectively. Although the deltaG(H20) at the maximum stability of both proteins is around 16 kcal/mol, Concanavalin A exhibits greater stability at higher temperatures. The T(g) obtained for Concanavalin A and WBA II were 21 degrees C apart at 87.2 and 66.6 degrees C, respectively. The higher conformational stability at higher temperatures and the T(g) of Concanavalin A as compared to that of WBA II are largely due to substantial differences in the degree of subunit contact in these dimeric proteins. Ionic interactions and hydrogen bonding between the monomers of the two proteins also seem to play a significant role in the observed stability differences between these two proteins.

Quaternary association and reactivation of dimeric concanavalin A

“…The ANS fluorescence data thus demonstrate a biphasic denaturation pathway of dimeric ConA, which is similar to those observed in the equilibrium unfolding of tetrameric ConA [26]. It may be mentioned that the equilibrium unfolding of dimeric ConA in guanidine hydrochloride follows a two-state process as reported [24].…”

“…The three-state equilibrium denaturation of dimeric ConA in urea has been analyzed thermodynamically in a similar manner as described [26]. The total free energy for dissociation (N 2 ↔ 2N) and subsequent unfolding (N ↔ U) reactions of dimeric ConA is obtained as 16.3 kcal mol −1 (Table 1), which is slightly higher than the value of 14.7 kcal mol −1 reported for guanidine hydrochloride-induced unfolding of dimeric ConA as a two-state process [24]. A comparison of thermodynamic parameters for dimeric and tetrameric ConA (Table 1) points out a remarkable difference in the free energy of dissociation ( G dis,aq ).…”

“…In the past few years, reports on reversible unfolding of several legume lectins have appeared in the literature [23,24]. Recently, we have demonstrated that the equilibrium unfolding reactions of soybean agglutinin and tetrameric ConA involve the formation of a structured monomeric intermediate [25,26].…”

X-ray structure of a galactose-specific lectin from Spatholobous parviflorous

“…The jelly roll model is characterized by the 6-stranded anti-parallel ␤-sheet, a curved 7-stranded front ␤-sheet and a short 5-stranded ␤-sheet to complete the appearance of the molecule as jelly roll. The sheets are connected by many loops [36]. A little over 50% of the residues belong to the loops connecting these sheets or the strands in them.…”

Differing structural characteristics of molten globule intermediate of peanut lectin in urea and guanidine-HCl

“…The unfolding pathways of many legume lectins in presence of these denaturants have been shown to be either a simple two-state (monophasic) or a multi-state (multiphasic) involving structured or partially folded intermediates [9][10][11][12][13][14][15][16][17]. Recently, we have explored the distinguishing structural aspects of soybean agglutinin and concanavalin A in different states that appear in their unfolding/refolding pathways [18,19], and demonstrated a unique molten globule fragment chain of pea lectin in its unfolding reaction in GdnHCl [20].…”