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J. Biol. Chem., Vol. 281, Issue 52, 40420-40428, December 29, 2006

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Cryoelectron Microscopy and EPR Analysis of Engineered Symmetric and Polydisperse Hsp16.5 Assemblies Reveals Determinants of Polydispersity and Substrate Binding^*

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

We have identified sequence and structural determinants of oligomer size, symmetry, and polydispersity in the small heat shock protein super family. Using an insertion mutagenesis strategy that mimics evolutionary sequence divergence, we induced the ordered oligomer of Methanococcus jannaschii Hsp16.5 to transition to either expanded symmetric or polydisperse assemblies. A hybrid approach combining spin labeling EPR and cryoelectron microscopy imaging at 10Å resolution reveals that the underlying plasticity is mediated by a packing interface with minimal contacts and a flexible C-terminal tether between dimers. Twenty-four dimeric building blocks related by octahedral symmetry assemble into the expanded symmetric oligomer. In contrast, the polydisperse variant has an ordered dimeric building block that heterogeneously packs to yield oligomers of various sizes. Increased exposure of the N-terminal region in the Hsp16.5 variants correlates with enhanced binding to destabilized mutants of T4 lysozyme, whereas deletion of this region reduces binding. Transition to larger intermediates with enhanced substrate binding capacity has been observed in other small heat shock proteins including lens -crystallin mutants linked to congenital cataract. Together, these results provide a mechanistic perspective on substrate recognition and binding by the small heat shock protein superfamily.

Received for publication, August 31, 2006 , and in revised form, October 26, 2006.

^* This work was supported by NEI, National Institutes of Health Grant R01-EY12018 (to H. S. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and supplemental Tables S1 and S2.

¹ These authors contributed equally to this work.

² To whom correspondence may be addressed: Dept. of Molecular Physiology and Biophysics, Vanderbilt University, 2215 Garland Ave., 741 Light Hall, Nashville, TN 37232. Tel.: 615-322-3307; Fax: 615-322-7236; E-mail: Hassane.mchaourab{at}vanderbilt.edu.

³ To whom correspondence may be addressed: Dept. of Molecular Physiology and Biophysics, Vanderbilt University, 2215 Garland Ave., 710 Light Hall, Nashville, TN 37232. Tel.: 615-322-7908; Fax: 615-322-7236; E-mail: phoebe.stewart{at}vanderbilt.edu.

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