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J. Biol. Chem., Vol. 279, Issue 16, 16425-16432, April 16, 2004

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Binding of Destabilized B2-Crystallin Mutants to -Crystallin

THE ROLE OF A FOLDING INTERMEDIATE^*

Hasige A. Sathish, Hanane A. Koteiche, and Hassane S. Mchaourab

From the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232

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 -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 -crystallin and B2-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 -crystallin is detected through changes in the emission properties of a bimane fluorescent probe site-specifically introduced at a solvent exposed site in B2-crystallin. -Crystallin binds the various B2-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 -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 -crystallin to detect subtle changes in the population of B2-crystallin excited states supports a central role for this chaperone in delaying aggregation and scattering in the lens.

Received for publication, December 8, 2003 , and in revised form, January 28, 2004.

^* This work was supported by NEI, National Institutes of Health Grant EY-R0112018. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 615-322-3307; Fax: 615-322-7236; E-mail: hassane.mchaourab{at}vanderbilt.edu.

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