Unfolding and refolding studies of frutalin, a tetrameric d-galactose binding lectin

Abstract

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ : mol 21 and 12.34 kJ : mol 21 , in the presence and absence of D-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by sizeexclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.Keywords: Artocarpus incisa lectin; frutalin; lectin refolding; lectin unfolding; protein refolding.Our current understanding of the protein folding mechanism is the result of intense studies using both theoretical and experimental biophysical methods. This complex problem concerning the mechanism by which proteins adopt one specific fold among those possible, has been experimentally investigated recently [1 -3]. Understanding this mechanism would provide a powerful tool for drug design and for comprehension of cellular organization at the molecular level. The fact that proteins with different sequences adopt the same fold suggests that the number of folding pathways is limited, probably, to a few hundred [4]. The b sheet class of proteins has been poorly represented in folding studies [5], even though they are thought to be important in the understanding of the formation of the b sheet that differs from the folding properties of helical and mixed a/b proteins. In recent years, the participation of abnormal b sheet structures in Alzheimer's, Huntington's and prion diseases has been demonstrated [6]. On the other hand, this class of b sheet proteins contains families whose members show high structural homology and sequential identity, although with different levels of specificity and affinity for specific binding [7,8]. Some of these b sheet proteins are the lectins, a particular carbohydrate-binding protein class widely distributed in all life forms that can mediate several biological events such as the recognition of molecules present in membranes or in the extracellular matrix [9].We have des...

Folding and Homodimerization of Wheat Germ Agglutinin

“…An exception to this is the case of frutalin, which shows a DG A of only À6 kcal mol À1 (dimer units). Nonetheless, the actual stability of frutalin corresponds to that of the tetramer, as this oligomer forms from the unfolded free subunits in an all-or-none process (39). In fact, this formation mechanism and high structural stability are not surprising for proteins that require the oligomeric state to perform their biological activity.…”

Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

“…The fluorescence emission spectrum of the purified recombinant frutalin showed an emission maximum at 335 nm. Native frutalin presents a fluorescence emission maximum at 333 nm [7].…”

Involvement of phosphatidylinositol-3 kinase–Akt and nuclear factor kappa-B pathways in the effect of frutalin on human lymphocyte

“…Protein concentration was determined by Bradford's method and the purity of frutalin in solution was confirmed by the presence of only two bands (~15.5 kDa and~12 kDa) after SDS-PAGE. The hemagglutinating effect of frutalin against human erythrocytes (O group) was determined as described [20] and the lectin showed hemagglutinating on activity at 0.2 Ag/ml (minimum dose capable of agglutinating 2% erythrocyte suspension).…”

“…Frutalin, an a-d-galactose-binding lectin purified from Artocarpus incisa seeds, shares many structural characteristics with jacalin [19]. Physical-chemical and biological studies have shown that frutalin possessed presented hemagglutinating activity greater than jacalin [20]. More recently, we have demonstrated that interaction of frutalin with human neutrophils induced chemotaxis and triggered the oxidative burst.…”