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J. Biol. Chem., Vol. 281, Issue 41, 30782-30793, October 13, 2006

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Glutamine Deamidation Destabilizes Human D-Crystallin and Lowers the Kinetic Barrier to Unfolding^*

From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human D-crystallin (HD-Crys) is a two-domain -sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 °C, or urea at pH 3.0, 20 °C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.

Received for publication, April 24, 2006 , and in revised form, July 28, 2006.

^* This work was supported in part by National Institutes of Health Grant GM17980 (to J. K.). 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.

¹ Supported by a Cleo and Paul Schimmel graduate fellowship.

² Supported by a United Negro College Fund-Merck graduate fellowship.

³ To whom correspondence should be addressed: Dept. of Biology, Massachusetts Institute of Technology, 31 Ames St., Rm. 68-330, Cambridge, MA 02139. Tel.: 617-253-4700; Fax: 617-252-1843; E-mail: jaking{at}mit.edu.

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