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 characterizatio n. Here we describe a solution to this problem for diacylglycerol kinase (D GK), an integral membrane protein from Escherichia coli. Twelve enhanced st ability mutants of DGK were obtained using a simple screen. Four of the mut ations were combined to create a quadruple mutant that had improved stabili ty in a wide range of detergents. In n-octylglucoside, the wild-type DGK ha d a thermal inactivation half-life of 6 min at 55 degrees C, while the quad ruple mutant displayed a half-life of 35 min at 80 degrees C. In addition, the quadruple mutant had improved thermodynamic stability. Our approach sho uld be applicable to other membrane proteins that can be conveniently assay ed.