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Inactivation mechanism of the membrane protein diacylglycerol kinase in detergent solution

Department of Chemistry and Biochemistry and the UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Molecular Biology Institute, UCLA, Los Angeles, California 90095-1570, USA

Reprint requests to: James U. Bowie; Department of Chemistry and Biochemistry, UCLA, Molecular Biology Institute, 611 Charles E. Young Dr. E., Los Angeles, California 90095-1570, USA; e-mail: bowie{at}mbi.ucla.edu; fax: (310) 206-4749.

We have examined the irreversible inactivation mechanism of the membrane protein diacylglycerol kinase in the detergents n-octyl-ß-D-glucopyranoside (OG) at 55°C and n-decyl-maltopyranoside (DM) at 80°C. Under no inactivation conditions did we find any direct evidence for the chemical modifications that are commonly found in soluble proteins. Moreover, protein inactivated at 55°C in OG could be reactivated by an unfolding and refolding protocol, suggesting that the protein is inactivated by a stable conformational change, not a covalent modification. We also found that the inactivation rate decreased with both increasing protein concentration and increasing thermodynamic stability, consistent with an inactivation pathway involving transient dissociation and/or unfolding of the protein. Our results suggest that the primary cause of diacylglycerol kinase inactivation is not low solubility, but poor intrinsic stability in the detergent environment.

Keywords: Stability; irreversible inactivation; denaturation; aggregation; half life

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