Monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck δ2-crystallin

Abstract

Duck δ2-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 μM 3. Dimers were also detected during the dissociation and refolding processes. The sharp enhancement of 1-anilino naphthalene-8-sulfonic acid (ANS) fluorescence at 1M GdnHCl strongly suggested that the dissociated monomers were in a molten globule state under these conditions. The similar binding affinity (≈ 60 μM) 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 δ 2-crystallin unfolding.