Thermal Denaturation of AQP 0 and Protection by Alpha Crystallin

Abstract

Purpose:: Alpha crystallin readily binds to the lipids and cytoskeletal elements of the lens membranes. Earlier studies suggest that AQP0 is not the primary binding site for alpha crystallin under ‘normal conditions.’ In this study we provide evidence that under thermal-stress conditions alpha crystallin specifically binds to AQP0. We also studied the kinetics of thermal aggregation and the effect of thermal stress on water permeability function of AQP0.

Methods:: Heat-induced protein denaturation of calf AQP0 was determined by measuring light scattering. Interaction of AQP0 with alpha crystallin was analysed by chromatographic and immunochemical methods. Protein structural changes were monitored by circular dichroism (CD). Water permeability function of AQP0 was measured using proteoliposomes by the stopped-flow apparatus.

Results:: There was a time- (at 50⁰C) and temperature-dependent (from 25⁰C - 60⁰C) increase in the aggregation of AQP0. The kinetics of aggregation was dependent on AQP0 concentration. At the same time higher concentrations of alpha crystallin (1:1 ratio of AQP0 and alpha crystallin, w/w) completely prevented thermal aggregation of AQP0, whereas lower concentrations (10:1) resulted in slowing the rate of aggregation. HPLC analysis showed that alpha crystallin binds irreversibly to AQP0. Immunoaffinity precipitation confirmed the interaction of alpha crystallin and AQP0. CD data suggested secondary structural changes of AQP0 during thermal denaturation and alpha crystallin prevented the loss of the secondary structures. There was a significant difference in the water permeability function of AQP0 due to thermal denaturation, which was restored by the addition of alpha crystallin.

Conclusions:: Alpha crystallin binds to AQP0 when the latter is undergoing thermal denaturation. Alpha crystallin seems to form two kinds of associations with AQP0, the static- and dynamic-interactions. During static interaction alpha crystallin binds tightly to AQP0 and does not allow the latter to denature even at higher temperatures. At lower concentrations, alpha crystallin interacts with AQP0 in a dynamic manner, which slows down the process of aggregation. Taken together these studies indicate that under normal conditions AQP0 may not be a binding site for alpha crystallin, but under thermal stress alpha crystallin readily binds to AQP0 to protect the latter from denaturation.

Keywords: chaperones • protein structure/function • cataract