Kinetic Refolding Barrier of Guanidinium Chloride Denatured Goose δ-Crystallin Leads to Regular Aggregate Formation

Abstract

Delta-crystallin is the major soluble protein in avian eye lenses with a structural role in light scattering. Dissociation and unfolding of the tetrameric protein in guanidinium chloride (GdmCl) can be sensitively monitored by the intrinsic tryptophan fluorescence. In this study refolding of GdmCl-denatured delta-crystallin was investigated. A marked hysteresis was observed while refolding by dilution of the 5 M GdmCl-denatured delta-crystallin. The secondary structure of the refolded protein was largely restored. However, monitoring intrinsic fluorescence of single tryptophan mutants indicated that the microenvironment of domain 1 (W74) was not restored. The region containing W169, which is close to the dimer interface, remained exposed following refolding. During refolding of the wild-type protein, dimeric, tetrameric, and aggregate forms were identified. The ratio of tetramer to dimer increased with time, as judged by gel-filtration chromatography and nondenaturing gel electrophoresis. However the observed levels of tetramer did not return to the same levels as observed before GdmCl treatment. The proportion of tetramer was significantly decreased in the N-25 deletion mutant and it did not increase with time. These results suggest that there is a kinetic barrier for assembly of dimers into tetramers. The consequence of this is that dimers refold to form aggregates. Aggregation seems to follow a nucleation mechanism with an apparent reaction order of 4.7+/-0.2, suggesting four or five monomers constitute the core structure of nucleus, which propagate to form high molecular weight aggregates. Addition of alpha-crystallin during refolding prevents aggregation. Thioflavin T and Congo red assays indicated a regular structure for the protein aggregates, which appear as hollow tubules packed into helical bundles. Aggregate formation was protein concentration dependent that progressed via two stages with rate constants of 0.0039+/-0.0006 and 0.00043+/-0.00003 s(-1), respectively. We propose that the N-terminal segment of delta-crystallin plays a critical role in proper double dimer assembly and also in the assembly of nucleus to aggregate formation.

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

“…This contrasts with the slow refolding of GdmCl denatured wild-type protein into its tetrameric form with no detectable activity. The slow recovery of the quaternary structure for the latter protein is due to an energy barrier for the appropriate assembly of double dimers, as reported previously [ 14 ]. The results suggest that the conformation of the denatured monomers which was unfolded by stepwise dissociation or directly unfolded with 5 M GdmCl could be different.…”

“…These dimers are unstable and they are prone to self-associate into protein aggregates, and this process competes with the formation of native tetramers [ 8 ]. Hence, the assembly of two dimers acts as a kinetic barrier in the refolding pathway [ 14 ]. The proper assembly of double dimers is thus important for producing a stable δ-crystallin quaternary structure.…”

“…When wild-type δ-crystallin was unfolded in GdmCl, the first transition for subunit dissociation resulted in a monomeric intermediate with a molten globule conformation [ 12 , 13 ]. During the refolding of 5 M GdmCl denatured δ-crystallin a marked hysteresis was observed, suggesting that the quaternary structure of that inappropriate assembled species might be related to the conformation of the refolded monomers [ 14 , 16 ]. The presence of stable intermediate during unfolding of K315A mutant protein in the presence of urea suggests that the interactions provided by this residue at the interfaces might provide an energy barrier for subunit dissociation.…”

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

“…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.…”

“…However, the final step, which is the assembly of dimers into tetramers, is a kinetic barrier in the refolding pathway. The consequence of this is that proteins associate into other stable conformations, leading to aggregate formation [13]. Identification of dissociated monomers in the presence of guanidinium hydrochloride indicates weak association interactions at the dimer–dimer interfaces.…”