This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. G-protein coupled receptors (GPCRs) represent a large group of sensory and nonsensory seven transmembrane (TM) helix receptors that require ligand-dependent activation to initiate heterotrimeric G-protein mediated intracellular signaling cascades. The structural basis for the interaction of a GPCR with its cognate G-protein, and the subsequent activation of the G-protein by R*, are not well understood. Thus, the overall goal of our research is to apply NMR methods to probe how structural signals from R* are propagated to the G-protein, resulting in guanine nucleotide exchange. To achieve thes goals, we are using a model system based on the signaling of the G-protein, transducin (Gt), by the light-activated GPCR, rhodopsin. This work is made possible by our ability to generate milligram quantities of isotope-labeled, full length G-protein ?-subunits that can be reconstituted with G-protein ??-subunits (G??) to form functional heterotrimeric G-proteins. In our analysis of the ?-subunit, we have observed a dramatic increase in the sensitivity in data acquired on a local 800 MHz NMR spectrometer when compared to a 600 MHz spectrometer. While we attribute the spectral improvements in some part to the general benefits of improved sensitivity and resolution afforded by the higher magnetic field, the higher magnetic field also appears to help us overcome intrinsic dynamics (CSX) in the protein backbone which severely compromised experiments taken at 600 MHz. Given the enabling success of our use of 800 MHz data for the purposes of assignment and analysis of the G-protein ?-subunit, we would like to collect 15N-HSQC, 15N-CPMG-HSQC and 3D15N-edited NOESY spectra at the NMRFAM on its 900 MHz spectrometer to see if any further improvements can be achieved by going to even higher field.
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