DETERMINING THE MOLECULAR ENVELOPE OF THE COMPLEX OF A G-PROTEIN AND ITS RECEPTO

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 G蛋白偶联受体是介导大多数细胞对激素、神经递质和感觉刺激的反应的7跨膜螺旋蛋白。与Brian Kobilka博士合作，我们一直在研究β 2-肾上腺素能受体的晶体结构，这是一种具有良好特征的G蛋白偶联受体。这些是配体激活的G蛋白偶联受体的第一个结构。迄今为止，该受体及相关受体的结构都处于失活状态，没有受体与G蛋白活性复合物的结构。一个关键的问题是如何激活配体稳定的受体，可以与它的同源异源三聚体G蛋白和触发核苷酸交换的构象。我们最近已经成功地生产了一个稳定的复合物的受体结合到一个激活配体和G蛋白。该复合物，约150 kDa，是单分散的，通过尺寸排阻色谱法和电子显微镜测定。我们提出的解决方案X射线散射测量，以评估我们是否可以获得有关的复杂的整体形状的信息，因为我们有两个单独的组件的晶体结构。受体和G蛋白之间的化学计量的确定将是非常重要的，因为有一些矛盾的结果在复合物中的受体的寡聚化性质。我们还想应用溶液散射来研究配体和拮抗剂/激动剂在复合物的整体结构中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. G-protein coupled receptors are 7-transmembrane helix proteins that mediate the majority of cellular responses to hormones, neurotransmitters, and sensory stimuli. In collaboration with Dr. Brian Kobilka, we have been studying crystal structures of the beta2-adrenergic receptor, a well-characterized G-protein coupled receptor. These were the first structures of a ligand-activated G protein coupled receptor. Thus far all structures of this and related receptors have been of the inactive state, and there are no structures of the active complex of receptor and G protein. A key question is how activating ligands stabilize the conformation of the receptor that can interact with its cognate heterotrimeric G protein and trigger nucleotide exchange. We have recently succeeded in producing a stable complex of the receptor bound to an activating ligand and a G protein. The complex, approximately 150kDa, is monodisperse as determined by size exclusion chromatography and electron microscopy. We propose solution x-ray scattering measurements to assess if we can obtain information about the overall shape of the complex, since we have the crystal structures of the two individual components. The determination of the stoichiometry between the receptor and G protein would be of significant importance as there are a number of conflicting results on oligomerization property of the receptor in the complex. We would also like to apply solution scattering to study effects of ligand and antagonists/agonists in the overall structure of the complex.