Abnormalities, such as single point mutations, in human γD-crystallin proteins have been implicated in cataracts, a condition where aggregation and phase separation of crystallin proteins in the eye lens leads to partial or total blindness due to lens cloudiness. We seek to measure changes in the solution behavior of bovine γB-crystallin using nuclear magnetic resonance (NMR) spectroscopy. A structural alignment using DALI reveals that bovine γB-crystallin and human γD-crystallins share 75% sequence identity over 171 amino acids and have an RMSD of 0.6 Å. A series of 15N T1 and T2 relaxation experiments and DOSY diffusion experiments have been conducted on γB-crystallin to better understand the effects of temperature and protein concentration on the rotational and translational diffusion of protein molecules, protein-protein attractions, and potential protein complexes at low to intermediate concentrations. Multiple repetitions of the experiments have allowed us to optimize our methodology. Preliminary results show that rotational diffusion slows down with increasing concentrations of up to 2-3 millimolar protein more rapidly than can be rationalized by viscosity changes, thereby giving a novel quantitative indicator of attractions between γB-crystallins. The corresponding temperature dependence of rotational diffusion is consistent with that expected from buffer viscosity vs. temperature.

Effect of temperature and concentration on the diffusion of a lens protein, γB-crystallin

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.