Real Time NMR Folding Study of the Human Gamma D Crystallin in the Presence of Metal Ions

Abstract

Cataract is a leading cause of blindness worldwide, and is due to aggregation of eye lens proteins. Age, UV radiation, oxidation, metal ions, deamidation, and truncations may be cause covalent protein damage that induces aggregation. In particular, metal ions have been implicated as a potential etiological agent for cataract. Human γD (HγD) crystallin is one of the most abundant crystallins in the lens nucleus and its non-amyloid aggregation is associated with cataracts. HγD is a highly stable protein with two homologous domains (N-terminal and C-terminal), each containing two Greek key motifs forming a β-sandwich of eight intercalated β-strands. Domain swapping from a perturbed conformation has been proposed as the mechanism of aggregation. Micromolar concentrations of Cu(II) and Zn(II) ions specifically induces the aggregation of this stable protein. In this work, NMR (H-N-HSQC) experiments were performed to study the interaction of Cu(II) and Zn(II) ions with HγD and identify possible specific metal binding sites in the protein. Moreover, we have characterized the unfolding of HγD in the presence of these ions with atomic resolution using real-time NMR spectroscopy. These studies provide further insight into the metal ion coordination properties of HγD, and the role of Cu(II) and Zn(II) in its non-amyloid aggregation. This research has been supported by: MIT-Seed Funds and NIH EY015834.