An alternative structural isoform in amyloid-like aggregates formed from thermally denatured human γD-crystallin.

The eye lens protein γD-crystallin contributes to cataract formation in the lens. In vitro experiments show that γD-crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV-B photodamage results in its C-terminal domain forming the β-sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet-like aggregates that contain cross-linked oligomers of the protein, according to transmission electron microscopy and SDS-PAGE. We use two-dimensional infrared spectroscopy to show that these aggregates have an amyloid-like secondary structure with extended β-sheets, and use isotope dilution experiments to show that each protein contributes approximately one β-strand to each β-sheet in the aggregates. Using segmental (13) C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N-terminal and C-terminal domains contribute to β-sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N-terminal domain into the fiber structure to intermolecular cross linking.