The determination of protein structures is a vital component of our understanding of nature. In the area of medicine and drug design the structure of the protein can greatly facilitate rational design of effective pharmaceuticals. Approximately half of current drug targets are integral membrane proteins, yet as the number of known structures approaches 50,000 only a few hundred of those are of integral membrane proteins, leaving a significant void in the data stream. It is clear that integral membrane proteins offer unique challenges to current methods of structure determination, and as yet there is no consensus approach for working with this especially difficult class of proteins. For NMR spectroscopy the dynamic nature observed for membrane proteins poses less of a sample preparation challenge than for other techniques such as X-ray crystallography. The limitation, however, for NMR has been the slow tumbling problem of large constructs such as integral membrane proteins. Our approach is to utilize NMR spectroscopy to take advantage of the ease of sample preparation of dynamic proteins, and uses a unique approach to overcome the slow tumbling problem. It is based on our earlier work using reverse micelle encapsulation of proteins. In that approach, the protein of interest is encapsulated within the protective aqueous core of a reverse micelle particle and the entire assembly is dissolved in a low viscosity fluid such as liquid ethane. In the low viscosity fluid, the reverse micelle particle tumbles faster than the protein dissolved in bulk water. This provides a significant improvement in the NMR relaxation properties governing the efficiency of the modern """"""""triple resonance"""""""" experiments. The method allows high performance NMR spectra to be obtained on soluble proteins as large as 100 kDa without benefit of deuteration or the TROSY effect. Here we propose to adapt this approach to studies of integral membrane proteins by employing two recent breakthroughs critical to sample preparation. We have developed a method of encapsulation that can be readily incorporated into an efficient, reliable and cost-effective apparatus for the preparation of samples for NMR spectroscopy. The prototype instrument will be built as part of this Phase I project. This avenue of research will demonstrate that not only is encapsulating integral membrane proteins viable, but can be done with sufficient through-put that it becomes meaningful as a structure determination tool as well as biochemical assay platform. To improve the robustness of the method we propose to explore and expand the available surfactant matrix space using the KcsA potassium channel, a homotetrameric helical bundle, as a model system. By rapidly-screening the effects of a variety of surfactant combinations we expect to be able to develop predictive encapsulation strategies that can be applied to new systems. Finally, we will take on the challenge of encapsulating the ?2-adrenergic GPCR and show conformational specificity of the protein by NMR spectroscopy. These studies should establish the reverse micelle solubilization method as general approach to structural studies of integral membrane proteins.
Approximately half of existing pharmaceuticals on the market target integral membrane proteins. Of these proteins very few have been studied structurally at the atomic level. The demand for high resolution structures for developing a detailed understanding of the molecular basis for life and for disease requires tools capable of delivering molecular level structural information. This proposal seeks to continue the development of a novel approach to structure determination by nuclear magnetic resonance. If successful, this technology could serve as a powerful platform for the rational design of pharmaceuticals for the treatment of an array of human diseases.
