ACQUISITION OF A 3D15N-EDITED NOESY FROM A G-PROTEIN ALPHA-SUBUNIT AT 900 MHZ

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 G蛋白偶联受体（GPCR）代表一大组感觉和非感觉七跨膜（TM）螺旋受体，其需要配体依赖性激活以启动异源三聚体G蛋白介导的细胞内信号级联。 GPCR与其同源G蛋白相互作用的结构基础以及随后通过R* 激活G蛋白的结构基础尚未得到很好的理解。 因此，我们研究的总体目标是应用NMR方法来探测R* 的结构信号如何传播到G蛋白，从而导致鸟嘌呤核苷酸交换。 为了实现这些目标，我们正在使用一个模型系统的基础上的信号G蛋白，转导（Gt），通过光激活的GPCR，视紫红质。 这项工作是可能的，因为我们有能力产生毫克量的同位素标记的全长G蛋白亚基，这些亚基可以与G蛋白亚基（G）重组，形成功能性异源三聚体G蛋白。 在我们的分析中，- 亚基，我们已经观察到一个显着增加的灵敏度在本地800 MHz的NMR光谱仪上获得的数据相比，600 MHz的光谱仪。 虽然我们将光谱的改善部分归因于更高磁场所提供的灵敏度和分辨率提高的一般好处，但更高的磁场似乎也有助于我们克服蛋白质骨架中的内在动力学（CSX），这严重损害了在600 MHz下进行的实验。 鉴于我们成功地使用800 MHz数据进行G蛋白亚基的分配和分析，我们想在NMRFAM的900 MHz光谱仪上收集15 N-HSQC，15 N-CPMG-HSQC和3D 15 N编辑的NOESY光谱，看看是否可以通过更高的场来实现任何进一步的改进。

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.