Insights into the structure and aggregation of lens crystallins and other aggregation-prone proteins

Abstract

Cataract is the world's leading cause of blindness. The destabilization, partial unfolding, and aggregation of lens crystallin proteins cause the loss of lens transparency (opacification) and cataract formation. Numerous congenital mutations and age-related changes to the long-lived alpha-, beta- and gamma-crystallins are associated with cataract and their study has provided insight into the molecular basis of this disease. In this thesis, alpha- and gamma-crystallin isoforms have been characterised under crowded and oxidative conditions, respectively. Finally, the conformational heterogeneity of model proteins was studied by capillary electrophoresis as a prelude to such studies on the more complex crystallins. Chapter 2 details the structural characterisation of the disulfide-linked gammaS-crystallin dimer, an oxidative product in the aging lens. X-ray crystallography revealed an intermolecular disulfide bond from C24-C24' and two intramolecular disulfides, one in each subunit, between C22 and C26. Small angle X-ray scattering confirmed the extended in-solution biological assembly in lieu of a compact state. It was demonstrated that the disulfide-linked dimer was stable at glutathione concentrations akin to those in aged and catractous lenses. The dimer had a higher aggregation propensity compared to the monomeric form owing to uncooperative domain unfolding. These findings provide novel insight into the contributions of oxidative modification to the formation of age-related cataract. Chapter 3 describes the impacts that a highly crowded environment comparable to the eye lens has on the structure and function of the molecular chaperone alphaB-crystallin. Macromolecular crowding using Ficoll 400 induces significant destabilisation, unfolding, an increase in size/oligomeric state, and a loss of chaperone function leading to kinetically distinct amorphous and fibrillar aggregation. These results are recapitulated in-principle using the biologically relevant crowding agent bovine gamma-crystallin. Aggregation is prevented by the lens partner protein alphaA-crystallin at physiologically relevant ratios through an increase in the alphaA/alphaB-crystallin complex stability. These results complement multiple dilute in vitro and in vivo studies and provide support for therapeutic approaches prevent and reverse cataract via alpha-crystallin stabilisation. Chapter 4 investigates capillary electrophoresis as a method for studying the conformational heterogeneity of a protein. Bovine serum albumin (BSA), yeast alcohol dehydrogenase (YADH), and bovine alpha-lactalbumin (BLA) were used to assess the application of this method towards various conformational aspects in comparison to SEC-MALS. The method distinguished between BSA oligomers and two different monomer populations, multiple YADH monomer and tetramer conformations, and apo- and holo-BLA. The 'dispersity of electrophoretic mobilities' allowed a relative comparison of the levels of conformational heterogeneity between unrelated proteins. This enables for better interpretation of the heterogeneity of more complex proteins such as post-translationally modified crystallins in vivo and oligomeric alpha-crystallin. Overall, this thesis provides new insights into the molecular basis for post-translational and environmental changes in alpha- and gamma-crystallins that cause cataract. Show more