Applications of Two-Dimensional Infrared Spectroscopy to Studies of Structure and Mechanism in Lens Crystallin Protein Aggregates

Cataracts are a disease that involves protein aggregation in the lens that blurs vision and eventually leads to blindness. It is known that denaturation, often resulting from various post-translational modifications, can cause the misfolding of crystallin proteins, which are found in the insoluble extracts of cataractous lenses. Although the causes of crystallin misfolding have been identified, its mechanism is still largely unknown. Using two-dimensional infrared (2D IR) spectroscopy we have been able to provide insights into the aggregate structures and misfolding mechanisms that gammaD-Crystallin undergoes when treated with UV irradiation, heat or acid, with the aid of 13C domain-labeling and native chemical ligation. 2D IR is sensitive to protein secondary structure and is capable of revealing coupling between different vibrational modes through cross-peaks. AlphaB-Crystallin is a structural protein that is known to assemble into oligomeric molecular chaperones that interact with misfolded or damaged proteins. Here, we demonstrate that the interaction between alphaB-Crystallin and misfolded gammaD-Crystallin can be observed using 13C labeling and 2D IR spectroscopy, based on cross-peaks between the proteins' amide one bands. Our study provides a new way of monitoring protein-protein interactions that will be valuable in the further study chaperone interactions with many other disease-related protein aggregates and their aggregation intermediates.