Abstract
δ-Crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. This protein is structurally assembled as double dimers. Lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces to interact with each other. This study found that wild-type protein had both dimers and monomers present in 2–4 M urea whilst only monomers of the K315A mutant were observed under the same conditions, as judged by sedimentation velocity analysis. The assembly of monomeric K315A mutant was reversible in contrast to wild-type protein. Molecular dynamics simulations showed that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. These results suggest the critical role of Lys-315 in stabilization of the diagonal dimer structure. Guanidinium hydrochloride (GdmCl) denatured wild-type or K315A mutant protein did not fold into functional protein. However, the urea dissociated monomers of K315A mutant protein in GdmCl were reversible folding through a multiple steps mechanism as measured by tryptophan and ANS fluorescence. Two partly unfolded intermediates were detected in the pathway. Refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. The formation of aggregates was not prevented by the addition of α-crystallin. These results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs.
Highlights
Introduction δCrystallin is a taxon-specific eye lens protein
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
Wild-type and K315A mutant δ-crystallin purified to near homogeneity were used for all analysis
Summary
Introduction δCrystallin is a taxon-specific eye lens protein. It is the major soluble protein in the eye lens of reptiles and birds and functions as a structural protein to maintain the refraction properties of the lens [1,2]. δ-Crystallin and argininosuccinate lyase (ASL) are homologous proteins. Crystallin is a taxon-specific eye lens protein It is the major soluble protein in the eye lens of reptiles and birds and functions as a structural protein to maintain the refraction properties of the lens [1,2]. Δ-Crystallin and argininosuccinate lyase (ASL) are homologous proteins. Δ-Crystallin and ASL share about 70% amino acid sequence identity and function as homotetramers, with four identical multi-subunit active sites [1,2,3,4,5,6]. The helices in domain 2 of each monomer associate to form a central helix bundle, comprising the core structure of the protein (Fig 1A) [4,5,7,8,9,10]. Salt bridges and hydrophobic interactions are the major forces which stabilize the quaternary structure of the protein
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