Aggregation of Crystallin Proteins

Cataracts are a common protein misfolding disease of the ocular lens, which affects approximately 50% of the population over the age of 65. This disease results from accumulated damage to lens Crystallin proteins, which destabilizes their folds and causes them to aggregrate, resulting in the blurring of vision. Currently, the only treatment for cataracts is invasive surgical extraction that is carried out in the advanced stages of the disease. As a result, there is much interest in understanding the cause of cataracts and the mechanism by which they form.

γD-crystallin has two similar domains, both are consistent with Greek Key motifs. Using expressed protein ligation, we uniformly 13C-label one of the two domains so that they can be individually resolved in a 2D IR spectrum, as can be seen in Figure 1.

Figure 1: γD-crystallin protein model and 2D IR spectra. Isotope labeling schemes are noted on the figure.

In order to understand the structures of lens Crystallin aggregates and their mechanisms of formation, we have studied γD-crystallin under a variety of conditions, including radiation, heat and a pH change with acid exposure. The morphologies of the amyloid fibers that form upon denaturation are dramatically different. When denatured with radiation, γD-crystallin forms globular structures with fibers extending off due to the drying process (Figure 2). When treated with heat, γD-crystallin forms amyloid like β-sheets that sometimes twist and fold (Figure 3A and B). When incubated with acid, it forms thin, curly fiber structures (Figure 3C).

Figure 2: TEM images of undamaged (A) and UV-denatured (B) γD-crystallin. Analysis of the diameters of the spheres and fibers are seen in (C-E). (F) shows the ThT kinetics of fiber formation induced by UV light.

Figure 3: TEM images of heat denatured (A and B) and acid denatured (C) γD-crystallin. SDS page of heat denatured γD-crystallin seen in (D).

The 2D IR spectra reveal more structural information into the formation of these amyloid fibers (Figure 4). We can see that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain unfolds into disordered structures. By using a combination of 2D IR, mass spectrometry, SDS-PAGE gel (Figure 3D) and THT fluorescence (Figure 2F), we can study the detailed structure of aggregates like in γD-crystallin, as well as many other proteins that have implications in diseases like diabetes and Alzheimer's.

Figure 4: 2D IR spectra of acid-induced amyloid fiber formation of γD-crystallin. The labeling scheme is the same as in Figure 1.