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

Abstract

δ‐Crystallin is the major structural protein in avian and reptilian eye lenses, and confers special refractive properties. The protein is a homotetramer arranged as a dimer of dimers. In the present study, the roles of the side chains of Glu267, Lys315, and Glu327, which provide hydrogen bonds at the double dimer interface, were investigated. Hydrophobic side chain substitution led to all mutant proteins having an unstable dimer interface. The E267L/E327L mutant had the greatest sensitivity to temperature, urea and guanidinium hydrochloride denaturation, and the most extensive exposure of hydrophobic patches, as judged by 1‐anilinonaphthalene‐8‐sulfonic acid fluorescence, CD, and tryptophan fluorescence. In contrast, the E267L/K315L/E327L mutant showed higher stability than the E267L/E327L mutant. Some level of the dissociated dimeric form was observed in the K315L mutant, but it was not observed for the K315A and E267L/K315L mutants. The E327L mutant was partially in the dissociated dimeric form, whereas the E267/E327L mutant was predominantly dissociated into dimers. In contrast, the triple mutant of E267L/K315L/E327L retained a tetrameric structure. In the presence of urea, a stable monomeric intermediate with higher stability than the wild type was identified for the K315A mutant. Disruption of interfacial interactions at Glu267 led to polymerization of partly unfolded intermediates in the presence of 3 m urea. However, these polymeric forms were not observed with combinations of the E267L mutation with other mutations. These results indicate that these hydrogen bonds, which are present at different contact surfaces in the dimer–dimer interface, perform distinct functions in double dimer assembly. The coordination of these interactions is critical for the stability and tetramer formation of δ‐crystallin.

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly

“…Unfolding of the two proteins in urea solution followed a three-state process, with the [D] 1/2 values in the first transition at 3.6 ± 0.1 and 1.7 ± 0.1 M urea, respectively ( Fig 2B ) [ 8 , 12 ]. The differences in the denaturant concentration required for the half transition clearly show the potency of GdmCl when disrupting of the conformation of δ-crystallin [ 11 ].…”

“…Fractions were analyzed by SDS-PAGE and protein concentrations determined by the method of Bradford [ 18 ]. Proteins possessing a C-terminal His 6 tag were purified on Ni affinity column (Chelating Sepharose FF, GE Healthcare) then desalted using a Sephadex G-25 column (26 mm x 12 cm) as previously reported [ 11 ].…”

“…The quaternary structure of δ-crystallin consists of a double dimer. The contact surface between the two dimers is smaller than the interface within the primary dimer of the structure [ 11 ]. Hydrogen bonding, salt bridges and hydrophobic interactions are the major forces which stabilize the quaternary structure of the protein.…”

“…The interactions of E327 with both K299 and R302 and the interaction of E267 with Y158 at the dimer interface were found to stabilize the quaternary structure of δ-crystallin in a cooperative manner. Mutations of the residues involved in both these interactions caused the majority of dimers to dissociate, whilst only partial dissociation was observed when these interactions were individually disrupted, as judged by sedimentation velocity experiments [ 11 ].…”

“…In contrast, the K315A mutant protein was relatively stable. This protein was unfolded into an intermediate with a stable conformation at 3 M urea [ 11 ]. This phenomenon was not observed for the K315L mutant protein, which was unfolded under the same conditions.…”