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

Abstract

Duck d 2 -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 lM 3 . Dimers were also detected during the dissociation and refolding processes. The sharp enhancement of 1-anilino naphthalene-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 (% 60 lM) 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 d 2 -crystallin unfolding.

Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligand of MD‐2

“…The binding of these dyes to a hydrophobic surface leads to an increase in their fluorescence intensity and to a blue shift of the maximum fluorescence, making the dyes useful for studying the internal structure, polarity and ligand binding properties of proteins (27,30,37,38). The affinity of ANS for partially folded states of proteins is in the 10 micromolar range (39), which is several orders of magnitude weaker than the affinity levels found in this report. High affinity of MD-2 for bis-ANS probably reflects a similarity in the structural pattern between LPS and bis-ANS, comprising a pair of separated negative charges and a hydrophobic patch (Fig.…”

Substitution of residues at the double dimer interface affects the stability and oligomerization of goose δ‐crystallin

“…Unlike for other mutants, an intermediate state was observed after the first unfolding transition when the K315A mutant was unfolded in the presence of urea. The first unfolding transition was assumed to be subunit dissociation of δ‐crystallin [11,12]. The intermediate was able to retain a stable conformation in 2–5 m urea, and was even more stable than observed for the wild‐type protein when urea concentrations exceeded 4 m (Fig.…”

“…These results also indicated the critical role played by Glu267 in the proper association of the two dimers to form the tetrameric δ‐crystallin or alternative off‐pathway aggregation. Polymerization of the E267L mutant in the presence of 3 m urea provides a model for regular aggregate formation of δ‐crystallin in the unfolding and refolding pathway [12,13]. No polymerization was seen for the E267L double or triple mutants, suggesting different conformations for the partly unfolded intermediates of the E267L mutant and its double and triple mutants.…”

“…Tetrameric δ‐crystallin is able to dissociate into a monomeric molten globule intermediate in the presence of guanidinium hydrochloride [11,12]. In this process, the dimeric form is transiently produced and is prone to form off‐pathway aggregates [6].…”

The effect of N-terminal truncation on double-dimer assembly of goose δ-crystallin

“…Each monomer contains a central 20-helix bundle at the core of the protein structure (domain 2), which closely associates in the tetrameric form. Hydrophobic interactions are the major forces involved in the subunit association [8][9][10]. The active site is located in a cleft between three different monomer subunits of the tetrameric protein.…”