Building a thermostable membrane protein.
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 degrees C, while the quadruple mutant displayed a half-life of 35 min at 80 degrees C. In addition, the quadruple mutant had improved thermodynamic stability. Our approach should be applicable to other membrane proteins that can be conveniently assayed.
Zhou
Y
Y
Department of Chemistry and Biochemistry, UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, UCLA, Los Angeles, California 90095, USA.
Bowie
J U
JU
D002384:Catalysis; D019852:Diacylglycerol Kinase / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D008565:Membrane Proteins / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding
D008565:Membrane Proteins; D019852:Diacylglycerol Kinase