Membrane proteins play a crucial role in many cellular and physiological processes as mediators of material and information transfer between cells and their environment, between compartments within cells, and between compartments comprising organ systems. Although it is clearly feasible to obtain membrane protein structures, the rate of soluble protein structure determination is outpacing that of membrane proteins so that there is a gap between our level of understanding of membrane proteins and their soluble counterparts. We propose to extend a form of indirect-detection magnetic resonance spectrometry known as High Frequency ENDOR (electron nuclear double resonance) for application to membrane protein structure determination. This structural method has been called ENDOR Crystallography and has been used primarily in molecular crystals. High Frequency (HF) ENDOR Crystallography is well-suited to the large size of many membrane proteins, the small size of their crystals, and their limited ability to diffract X-rays. It is largely free of non-protein background signals from lipids and detergents yet can yield atomic resolution structures including experimentally determined proton locations in the most interesting regions of many proteins. We propose to develop High Frequency ENDOR Crystallography for structural determination in membrane protein crystals through the following three specific aims: 1) Develop high-resolution ENDOR methods suitable for ENDOR Crystallography of membrane protein crystals for implementation at high frequency/field; 2) Construct and implement an optimized High Frequency ENDOR probe for convenient crystal loading and rotation, high sensitivity, and automated data collection; 3) Develop methods for automated peak assignment and analysis of experimentally measured HFENDOR crystallographic data from membrane proteins. ENDOR Crystallography data will be collected from several representative membrane protein crystals (Cytochrome c Oxidase, the bc1 complex and the b6f complex) and protein structural coordinates will be extracted from that data.
These specific aims bring together a number of advanced techniques in order to advance the state of the art in membrane protein structure determination. ? ? ? ?
