Transmembrane signaling is a fundamental step in a cell's ability to sense and respond to environmental signals, which is critical to processes ranging from bacterial chemotaxis to the coordinated function of cells in multicellular organisms. Ligand binding is thought to induce conformational changes or clustering to transmit the signal, but the mechanisms are ill-defined, in part because of the lack of structural information on the membrane-bound receptor proteins. The proposed research focuses on bacterial chemotaxis receptors (the related Asp and Ser receptors) as model systems for probing mechanisms of transmembrane signaling. The first goals are to map structural changes that occur in the periplasmic and transmembrane regions upon ligand binding--the initiation and propagation of the excitation signal. The approach is to integrate novel solid-state NMR techniques which measure selected distances with biochemical methods for targeting these measurements to the sites of interest. Site-specific isotopic labeling is achieved by using mutagenesis to introduce unique residues for subsequent labeling. Rotational resonance and REDOR are used to measure interhelical distances and how they change in response to ligand binding and methylation; additional measurements are designed to discriminate different types of motion. Application of a novel spin-diffusion technique also tests for piston motions of transmembrane helices. In the poorly understood cytoplasmic domain, distance measurements focus on testing coiled-coil models for the methylation region. Solution NMR will determine whether c-fragment dimers unfold during dissociation to monomers, to test the proposed inter to intramolecular coiled-coil model. Complementary solid-state NMR experiments probing receptor dynamics will identify dynamic regions and any changes in dynamics upon ligand binding or methylation. These experiments will directly measure proposed differences in structure and dynamics between signaling states. The proposed research provides a unique approach to obtaining direct information on molecular mechanisms of transmembrane signaling. In addition, the integrated biochemistry/NMR strategies developed for this study will be applicable to other important questions of membrane protein structure and function.
| Sferdean, Fe C; Weis, Robert M; Thompson, Lynmarie K (2012) Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms. Biochemistry 51:6920-31 |
| Fowler, Daniel J; Harris, Michael J; Thompson, Lynmarie K (2012) Heat management strategies for solid-state NMR of functional proteins. J Magn Reson 222:112-8 |
| Fowler, Daniel J; Weis, Robert M; Thompson, Lynmarie K (2010) Kinase-active signaling complexes of bacterial chemoreceptors do not contain proposed receptor-receptor contacts observed in crystal structures. Biochemistry 49:1425-34 |
| Fowler, Daniel J; Khalifah, Peter G; Thompson, Lynmarie K (2010) Design and characterization of a calixarene inclusion compound for calibration of long-range carbon-fluorine distance measurements by solid-state NMR. J Magn Reson 207:153-7 |
