We propose to crystallize and solve the structure of an integral membrane protein, diacylglycerol kinase from Escherichia coli (DGK). DGK catalyzes the conversion of diacylglycerol and ATP to phosphatidic acid and ADP. A high resolution structure would be important for many reasons. First, it would represent the first structure of an integral membrane enzyme that catalyzes a chemical conversion, providing a model for a whole class of poorly studied membrane enzymes involved in lipid signaling and lipid metabolism. Investigations of catalysis in the complex membrane environment could hold many surprises. Second, DGK is an excellent model system for learning about membrane protein structure. It is small (121 residues), it can be produced in large quantities, there is a good genetic system and the purified protein is well behaved. A structure would enable detailed probing of the determinants of membrane protein structure. Finally, it is an excellent antibiotic target and a high resolution structure would facilitate the design of specific inhibitors. Membrane protein crystallization presents daunting challenges, however, and generally requires years of sustained effort. We have been unable to crystallize the enzyme by standard methods and propose alternative strategies that could also be applicable to other membrane proteins.
The specific aims are: I. Crystallize mutant proteins. We have created two types of mutants for crystallization. (1) Highly active mutants with multiple sequence changes. These mutants introduce new potential crystal lattice contacts. (2) Mutants with enhanced stability. Since crystallization requires a well-behaved protein, mutants with improved stability are more likely to crystallize. II. Crystallize DGK with added polar domains. Because lattice contacts in membrane protein crystals are usually made by the polar domains, one strategy for improving the probability of obtaining crystals is to increase the polar domain surface area. We propose two strategies for increasing the polar mass of DGK. (1) Add antibody Fv fragments, specific for folded DGK. This strategy was already used to crystallize another membrane protein. (2) Add a ribonuclease S domain via the addition of S- peptide to DGK.
| Faham, Salem; Bowie, James U (2002) Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J Mol Biol 316:1-6 |
| Zhou, Y; Lau, F W; Nauli, S et al. (2001) Inactivation mechanism of the membrane protein diacylglycerol kinase in detergent solution. Protein Sci 10:378-83 |
| Bowie, J U (2001) Stabilizing membrane proteins. Curr Opin Struct Biol 11:397-402 |
| Zhou, Y; Bowie, J U (2000) Building a thermostable membrane protein. J Biol Chem 275:6975-9 |
