tailieunhanh - Báo cáo Y học: Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin

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. | Eur. J. Biochem. 269 753-758 2002 FEBS 2002 Unfolding and refolding studies of frutalin a tetrameric D-galactose binding lectin Patricia T. Campana1 Derminda I. Moraes1 Ana C. O. Monteiro-Moreira1 2 and Leila M. Beltramini1 1Instituto de Fisica de Sao Carlos Universidade de Sao Paulo Sao Carlos Brasil 2Departamento de Bioquimica e Biologia Molecular Universidade Federal do Ceara Fortaleza Brasil 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 kJ-mol-1 and kJ-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. Keywords Artocarpus incisa lectin frutalin lectin refolding lectin unfolding protein refolding. Our current understanding of the protein folding mechanism is the result of .