Temperature-dependent oligomerization in M-crystallin: lead or lag toward cataract, an NMR perspective

Abstract

The oligomerization and/or aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from abnormal disease states like Alzheimer's and Parkinson's disease to the production of inclusion bodies, stability, and delivery of protein drugs. In the case of eye-lens proteins, oligomerization is implicated in cataract formation. In the present study, we have investigated the temperature driven oligomerization of M-crystallin, a close homologue of eye-lens proteins, using NMR spectroscopy and dynamic-light scattering (DLS). The NMR data primarily included R₁, R₂ relaxation rates and nOes of the backbone amide groups recorded at three different temperatures, 25, 20, and 15°C. The major outcome of the study is the two fold increase in the overall tumbling time (τ_c) of M-crystallin on lowering the temperature from 25 to 15°C. An extrapolation of τ_c to a further lower temperature (5°C) may lead to a τ_c of ~19 ns that would correspond to a τ_c value of a tetrameric M-crystallin. These results also validate the observed changes in the hydrodynamic radius of M-crystallin, determined using DLS data. Further, the temperature-dependent protein dynamics of M-crystallin reveal considerable variation at/near the Ca²⁺-binding sites. A concerted analysis of the temperature dependent relaxation parameters and DLS data reveals that the self-association of the protein is not only a monomer-dimer equilibrium, but also goes to tetramers or other multimeric states. These higher states may co-exist in fast exchange with the monomeric and dimeric M-crystallin at milli-molar to sub-millimolar concentrations and at lower temperature.