Monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck delta2-crystallin.

Abstract

Duck delta2-crystallin is a soluble tetrameric lens protein. In the presence of guanidinium hydrochloride (GdnHCl), it undergoes stepwise dissociation and unfolding. Gel-filtration chromatography and sedimentation velocity analysis has demonstrated the dissociation of the tetramer protein to a monomeric intermediate with a dissociation constant of 0.34 microM3. Dimers were also detected during the dissociation and refolding processes. The sharp enhancement of 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence at 1 M GdnHCl strongly suggested that the dissociated monomers were in a molten globule state under these conditions. The similar binding affinity (approximately 60 microM) of ANS to protein in the presence or absence of GdnHCl suggested the potential assembly of crystallins via hydrophobic interactions, which might also produce off-pathway aggregates in higher protein concentrations. The dynamic quenching constant corresponding to GdnHCl concentration followed a multistate unfolding model implying that the solvent accessibility of tryptophans was a sensitive probe for analyzing delta2-crystallin unfolding.