Abstract 1365 Using Nuclear Magnetic Resonance Spectroscopy to Interpret the Effect of Relevant Mutations on gamma B Crystallin Structure and Interactions

Crystallins are the predominant structural proteins in the eye lens. The dysfunction of specific inter-protein interactions between the crystallins is known to cause the proteins to aggregate and phase-separate, causing the lens to move from clear to cloudy. Posttranslational mutations, some of which are the result of aging, UV damage, or congenital mutations, can lead to changes in the chemical and physical properties of the proteins, thus affecting inter-protein crystallin interactions and resulting in cloudiness called cataracts. Studying biologically relevant mutants of crystallin proteins, in comparison with wild-type (nonmutated) crystallin, will provide more information on how specific residues dictate the detailed interactions between proteins, which can lead to cataracts. Here, we describe our work with biologically relevant site-directed mutants (S130W and P23T) of bovine γB crystallin, a homologue to human γD crystallin. Alongside other biochemical and biophysical experiments, we used nuclear magnetic resonance (NMR) spectroscopy to determine how the mutations affected the interactions between the γB crystallin proteins. Preliminary results suggest that chemical shifts determined using NMR are highly sensitive to changes in intermolecular interactions. These studies will add to our understanding of how mutations in crystallins affect inter-protein interactions, protein aggregation, and cataract formation. This work was supported by the Rochester Institute of Technology.