title: Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly.
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.
authors.affiliation: Pharmacy Division, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan.|Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Chih-Wei
authors.initials: CW
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Hui-Chen
authors.initials: HC
authors.lastname: Lin
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Chi-Yuan
authors.initials: CY
authors.lastname: Chou
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Wei-Chuo
authors.initials: WC
authors.lastname: Kao
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Wei-Yuan
authors.initials: WY
authors.lastname: Chou
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Hwei-Jen
authors.initials: HJ
authors.lastname: Lee
mesh_terms: D000818:Animals; D005777:Geese; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D066329:Protein Aggregates; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D038207:delta-Crystallins / Q000737:chemistry*
chemical_list: D066329:Protein Aggregates; D021122:Protein Subunits; D038207:delta-Crystallins; D008239:Lysine