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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 G蛋白偶联受体(GPCRs)是一大类感觉性和非感觉性七种跨膜(TM)螺旋受体，需要配体依赖的激活来启动异源三聚体G蛋白介导的细胞内信号级联反应。GPCR与其同源G蛋白相互作用以及随后由R*激活G蛋白的结构基础尚不清楚。因此，我们研究的总体目标是应用核磁共振方法来探索来自R*的结构信号是如何传播到G-蛋白，导致鸟嘌呤核苷酸交换的。为了实现这些目标，我们正在使用一个基于光激活的G蛋白转导蛋白(GT)信号的模型系统，即视紫红质。这项工作之所以成为可能，是因为我们能够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，形成功能性的异源三聚体G蛋白。在我们对亚基的分析中，我们观察到与600 MHz光谱仪相比，在本地800 MHz核磁共振光谱仪上获得的数据的灵敏度有了显著的提高。虽然我们将光谱的改善在一定程度上归因于较高磁场提供的灵敏度和分辨率提高的一般好处，但较高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)，这严重影响了在600 MHz进行的实验。鉴于我们能够成功地使用800 MHz数据来指定和分析G蛋白亚基，我们希望在NMRFAM的900 MHz光谱仪上收集15N-HSQC、15N-CPMG-HSQC和3D15N编辑的NOESY光谱，看看是否可以通过进入更高的场来实现进一步的改进。

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.