Only 0.36% of the 18,412 proteins in the Protein Data Bank are integral membrane proteins, yet membrane proteins comprise approximately 25% of the genome and are linked to a multitude of human diseases. Although the isolation, crystallization and NMR solution structure determination of membrane-bound proteins is viewed as difficult, new techniques are emerging to solve the technical problems. We will combine our expertise in cloning, expression, purification, X-ray crystallography and NMR to focus on structure determinations of two very well characterized E. coli membrane-bound oxidoreductases: nitrate reductase A (NarGHI) and DMSO reductase (DmsABC). These integral membrane-bound bacterial enzymes are complex multi-subunit proteins which participate in energy conservation. In addition, we will extend our research to two novel membrane-bound oxidoreductases: YedYZ and YdhXU, identified by genome analysis, which we believe also participate in energy conservation and have properties commensurate with our aims. All contain an array of cofactors including b-type hemes, iron sulfur clusters and molybdopterin cofactors which are essential for energy conservation in all living organisms. We have shown that NarGHI is ideal for the investigation of membrane protein overproduction, purification and crystallization and we believe that DmsABC is also an excellent candidate for investigation based on our intensive studies of this enzyme. The individual subunits Narl, YedZ and YdhU are excellent candidates to develop emerging multidimensional NMR techniques. Together our team will develop techniques that will be applicable to a range of membrane-bound proteins.
Our specific aims are to: (i) complete the atomic resolution structure of NarGHI utilizing our existing crystals that diffract to 1.9Angstrom resolution and selenomethionine labeled protein crystals, (ii) to develop improved over-expression and purification protocols for DmsABC to produce quantities of protein which will be subjected to detergent exchange, crystallization trials and X-ray structure determination, (iii) to carry out large-scale purification of YedYZ and YdhXU to produce quantities of protein which will be subjected to detergent exchange, crystallization trials and X-ray structure determination and (iv) to develop multidimensional NMR methods to address the structure of heme-containing integral membrane subunits that serve both an anchor and quinol oxidase function.
| George, Graham N; Doonan, Christian J; Rothery, Richard A et al. (2007) X-ray absorption spectroscopic characterization of the molybdenum site of Escherichia coli dimethyl sulfoxide reductase. Inorg Chem 46:2-4 |
