Binding of Destabilized βB2-Crystallin Mutants to α-Crystallin

Age-related changes in protein-protein interactions in the lens play a critical role in the temporal evolution of its optical properties. In the relatively non-regenerating environment of the fiber cells, a critical determinant of these interactions is partial or global unfolding as a consequence of post-translational modifications or chemical damage to individual crystallins. One type of attractive force involves the recognition by α-crystallins of modified proteins prone to unfolding and aggregation. In this paper, we explore the energetic threshold and the structural determinants for the formation of a stable complex between α-crystallin and βB2-crystallin as a consequence of destabilizing mutations in the latter. The mutations were designed in the framework of a folding model that proposes the equilibrium population of a monomeric intermediate. Binding to α-crystallin is detected through changes in the emission properties of a bimane fluorescent probe site-specifically introduced at a solvent exposed site in βB2-crystallin. α-Crystallin binds the various βB2-crystallin mutants, although with a significantly lower affinity relative to destabilized T4 lysozyme mutants. The extent of binding, while reflective of the overall destabilization, is determined by the dynamic population of a folding intermediate. The existence of the intermediate is inferred from the biphasic bimane emission unfolding curve of a mutant designed to disrupt interactions at the dimer interface. The results of this paper are consistent with a model in which the interaction of α-crystallins with substrates is not solely triggered by an energetic threshold but also by the population of excited states even under favorable folding conditions. The ability of α-crystallin to detect subtle changes in the population of βB2-crystallin excited states supports a central role for this chaperone in delaying aggregation and scattering in the lens.