Probing the Kinetics of Unfolding and Aggregation of Human Gamma-D Crystallin at Low pH Using Fourier Transform Infrared Spectroscopy



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Cataracts are one of the foremost public health concerns society faces today. According to the World Health Organization, cataracts are the leading cause of blindness in the world. A cataract is the clouding of the eye's natural lens. Upon development of a cataract, light cannot penetrate the cloudy lens properly, causing blurred images to appear on the retina or no image at all. Cloudiness of the lens is in large part due to the aggregation of the lens proteins. These proteins consist primarily of alpha, beta, and gamma crystallins, which comprise 90% of the total protein content of the lens. These proteins are expressed early in life and must remain stable throughout a person s lifetime. Human gamma-D crystallin (HgD) is a 173 amino acid beta sheet protein consisting of an N-terminal domain and a C-terminal domain connected by a 6 amino acid linker peptide. In each domain there are two Greek key motifs. This highly symmetric protein is very thermostable (melting temperature greater than 75˚C). When HgD is unfolded in guanidinium then refolded by dilution of the denaturant, a portion of the protein aggregates. This aggregate is amorphous (not fibrous) in morphology and has similar secondary structure to the wild type protein. However, when HgD is unfolded under conditions of low pH, it aggregates to form amyloid-like fibrils. These dual pathways for aggregation make HgD a very interesting model system for studying the protein aggregation process, and determining the factors which direct the aggregation towards the amorphous or amyloid structures. Infrared spectroscopy (IR) can be used to distinguish between amyloid-like aggregates and native beta sheets; amyloid structures give two narrow amide I bands at ~1620 and 1680 cm-1, while native beta sheets typically give a broad band at ~1633 - 1640 cm-1. Thus, IR spectroscopy can be used to probe amyloid formation. In this study, we use time-resolved IR spectroscopy to monitor the aggregation of HgD at low pH (~2.0) and 37°C. We find that the formation of amyloid aggregates is very sensitive to initial conditions, and demonstrate that this pathway is in competition with another aggregation pathway.



cataracts, human gamma-D crystallin