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 kJAEmol )1 and 12.34 kJAEmol, in the presence and absence of D-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion 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][2][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 this is critical for a complete 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 ligands [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 described and studied s...

cDNA Cloning and Functional Expression of the α-d-Galactose-Binding Lectin Frutalin in Escherichia coli

“…It belongs to the jacalin-related lectins family, more precisely to the galactose-binding sub-family, as it presents high similarities in sequence, structure and sugar specificity with the jacalin lectin (the galactose-specific lectin of A. integrifolia seeds-jackfruit, the first member of this family to be identified) [5, 6, this work]. Frutalin also presents functional properties identical to jacalin; however, it has a more noticeable biological activity than the jackfruit lectin, as shown by its higher hemagglutination activity [7]. Previous studies have shown that frutalin plays an important role in many biomedical applications.…”

Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin fromArtocarpus incisaseeds

“…The lectin from A. incisa, which is named frutalin (FTL), has been isolated and characterized (Moreira et al, 1997), and its refolding process has been studied using thermal and chemical denaturation (Campana et al, 2002). It belongs to the jacalin lectin-related family (Moraceae family) and has an affinity for -d-galactose monosaccharides and complex carbohydrates that contain Gal 1-3 glycans (Moreira et al, 1997;Nobre et al, 2010).…”

Recombinant production of plant lectins in microbial systems for biomedical application – the frutalin case study

“…The β chain is not visible due to its low molecular weight (2.1 kDa; Oliveira et al, 2008). In its native form, FTL is a tetrameric molecule, consisting of four monomers bound by non-covalent linkages, each containing one β and one α chain, forming four sugar-binding sites, with a predominantly β sheet conformation (Moreira et al, 1998; Campana et al, 2002) and an apparent molecular mass of 48–49 kDa (Moreira et al, 1998; Oliveira et al, 2008). FTL is a robust protein as it is stable up to 60°C and very resistant to chemical denaturation (Campana et al, 2002).…”