title: 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 x mol(-1) and 12.34 kJ x mol(-1), 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.
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brasil.
authors.forename: Patricia T
authors.initials: PT
authors.lastname: Campana
authors.forename: Derminda I
authors.initials: DI
authors.lastname: Moraes
authors.forename: Ana C O
authors.initials: AC
authors.lastname: Monteiro-Moreira
authors.forename: Leila M
authors.initials: LM
authors.lastname: Beltramini
mesh_terms: D002942:Circular Dichroism; D005453:Fluorescence; D037161:Galectins; D019791:Guanidine / Q000737:chemistry; D006386:Hemagglutination Tests; D006388:Hemagglutinins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D017510:Protein Folding
chemical_list: D037161:Galectins; D006388:Hemagglutinins; D019791:Guanidine