Abstract
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 alpha-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 alpha-crystallin and betaB2-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 alpha-crystallin is detected through changes in the emission properties of a bimane fluorescent probe site-specifically introduced at a solvent exposed site in betaB2-crystallin. alpha-Crystallin binds the various betaB2-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 alpha-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 alpha-crystallin to detect subtle changes in the population of betaB2-crystallin excited states supports a central role for this chaperone in delaying aggregation and scattering in the lens.
Highlights
In the inner regions of the lens, transparency and refractivity are dependent on the stability, high solubility, and packing of three families of proteins, collectively referred to as crystallins [1,2,3]
Equilibrium sedimentation studies of A3-crystallin molecular weight were interpreted in terms of a reversible equilibrium involving a monomeric intermediate with a ␥-crystallin-like conformation [20]
The folding equilibrium of -crystallin can be written as shown in Equation 1, D a 2I a 2U
Summary
In the inner regions of the lens, transparency and refractivity are dependent on the stability, high solubility, and packing of three families of proteins, collectively referred to as crystallins [1,2,3]. In the low protein turnover environment of lens fiber cells, the -crystallins are subject to extensive modifications, either programmed or as a result of oxidative and other types of damage [12, 13, 23,24,25,26]. Regardless of their origin, these modifications are expected to shift the equilibrium between dimeric, monomeric, and unfolded -crystallin. In the context of a mechanistic study of ␣-crystallin chaperone activity, we have developed an equilibrium binding assay
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