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J Biol Chem, Vol. 275, Issue 10, 6975-6979, March 10, 2000

Building a Thermostable Membrane Protein*

Yufeng Zhou and James U. BowieDagger

From the Department of Chemistry and Biochemistry, UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, UCLA, Los Angeles, California 90095

The poor stability of membrane proteins in detergent solution is one of the main technical barriers to their structural and functional characterization. Here we describe a solution to this problem for diacylglycerol kinase (DGK), an integral membrane protein from Escherichia coli. Twelve enhanced stability mutants of DGK were obtained using a simple screen. Four of the mutations were combined to create a quadruple mutant that had improved stability in a wide range of detergents. In n-octylglucoside, the wild-type DGK had a thermal inactivation half-life of 6 min at 55 °C, while the quadruple mutant displayed a half-life of 35 min at 80 °C. In addition, the quadruple mutant had improved thermodynamic stability. Our approach should be applicable to other membrane proteins that can be conveniently assayed.

* This work was supported by Department of Energy Grant DEFC0387ER60615 and National Institutes of Health Grants GM47485-08 and GM59164-01.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, UCLA, 405 Hilgard Ave., Los Angeles, CA 90095. Tel.: 310-206-4747; Fax: 310-206-4749; E-mail: bowie@ewald.mbi. ucal.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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