The ultimate goal of the project is to further develop a novel approach that will hopefully allow the application of modern solution NMR techniques to large and membrane proteins. Solvated ubiquitin will be dissolved in butane, containing sodium dis (2-ethylhexyl) sulfosuccinate (AOT) as a surfactant, according to established methods in Dr. Wand's group. Complete resonance assignments will be obtained and structure calculations will be performed on ubiquitin encapsulated in the reverse micelle. This will test the feasibility of applying triple-resonance NMR techniques in reverse micelles, and the comparison of the structure obtained in the reverse micelle with the crystal structure will demonstrate and validate the approach as applied to a water-soluble protein. Next, we will attempt to adopt the approach to diacylglycerol kinase. Circular dichroism and fluorescence methods will be used to develop conditions for the encapsulation of this membrane protein in its functional form. Resonance assignment and structural elucidation of diacylglycerol kinase will be performed by triple-resonance NMR methods. If time allows, this approach may be applied to a ligand-receptor complex.
| Kielec, Joseph M; Valentine, Kathleen G; Babu, Charles R et al. (2009) Reverse micelles in integral membrane protein structural biology by solution NMR spectroscopy. Structure 17:345-51 |
