Mechanism of a Protein Sensor: The Chaperone Activity of –Crystallin

Abstract

Abstract: : Mechanism of a Stability Sensor: The Chaperone Activity of α–Crystallin Purpose:To develop a mechanistic understanding of the chaperone activity of α–crystallin and Hsp27, we carried out a systematic thermodynamic dissection of the recognition and binding of a model substrate. Methods: The thermodynamic representation of the chaperone–substrate system is a set of three coupled equilibria that includes the substrate folding equilibrium, the chaperone oligomeric equilibrium, and the equilibrium binding between the chaperone and the substrate. We have perturbed the two former equilibria and quantitatively determined the consequences on binding. The substrate is a set of T4 Lysozyme (T4L) mutants that bind under conditions that favor the folded state over the unfolded state by 10²–10⁴ fold. The concentration–dependent oligomer equilibrium of Hsp27 was perturbed by mutations that alter the relative stability of two major oligomeric states including phosphorylation–mimicking mutations that result in the dissociation to a small multimer over a wide range of concentrations. Complex formation is detected either by the changes in the spectroscopic properties of a paramagnetic or fluorescence group or by calorimetry. Results: α–crystallin and Hsp27 differentially recognize the T4L mutants binding the more destabilized one to a larger extent. Binding occurs through two modes, each characterized by different affinity and number of binding sites, and results in complexes of different hydrodynamic properties. The two binding modes appear to recognize distinct conformations of T4L. Correlation of binding isotherms with size–exclusion chromatography analysis of the Hsp27 oligomer equilibrium demonstrates that the multimer is the binding–competent state. Mutants of the Hsp27 phosphorylation mimic that reverse the reduction in oligomer size also reduce the extent of T4L binding. Conclusions: α–crystallin and Hsp27 are stability sensors that can distinguish between substrates that have similar structures in the native state. Their chaperone activity is regulated by a switch encoded at the level of the oligomeric structure. This switch is activated by temperature and phosphorylation which shift the oligomer equilibrium towards the binding competent state.

Keywords: crystallins • chaperones