Molecular Structure and Function of Crystallins

National Eye Institute

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Abstract

gamma-Crystallins are associated with cataract in both human and animal models. They may also have stress related roles in other eye tissues, notably retina. We have shown that they can play a role in stabilization of cytoskeleton in lens. g-Crystallins have highly unusual solution properties that fit them for high protein concentration environments. They have stable, tightly folded structures but can unfold to form amyloid like fibrils. We have studied unfolding/refolding in members of the gS-crystallin family from birds and mammals. We have identified an unexpected mode of dimerization in many g-crystallins that correlates with the magnitude and direction of molecular dipoles. AUC data with our collaborators show that gS-crystallins do dimerize at high concentrations similar to those found in the lens. We are also investigating how oxidation and other stresses contribute to crystallin unfolding and aggregation, We now have a crystal structure for a variant of mouse gS under oxidizing conditions. This remarkable structure appears to be an aggregation intermediate involving intra- and inter-molecular S-S bonds, strained domain swapping and progressive loss of secondary structure. This adds important insight into the processes that lead to cataract. Further experiments show that the octameric structure in the crystal forms by dimerization of a disulfide linked tetramer but that geometry does not allow full disulfide formation. Additional experiments using circular dichroism and other techniques suggest that multimerization precedes disulfide formation and loss of secondary structure. This shows that the loss of organized structure is enhanced by the processes of aggregation, suggesting a cascade effect in cataract. We have been able to obtain the octamer in solution by dissolving crystals. Several techniques, including Mass photometry (ISCAMS) and cross-linking confirm the crystal structure is preserved in solution. As predicted, we find that the octamer is highly prone to aggregation in solution, mimicking what can occur in cataract formation. Structure is deposited in PDB ID 7RJ0