G PROTEIN/RECEPTOR ASSOC--STRUCTURAL CHARACTERIZATION

我们计划表征G蛋白与其膜的结合 使用实验（包括核磁共振， （主要是转让的NOE）和理论（包括计算机 用于结构细化和分子建模的模拟）技术。 含有不同细胞质部分的肽的合成 受体已部分实现，并将完成大胆这一点 授予期。将以三种不同形式检查肽; 直链-环化（利用亚甲基连接基以保持 12末端之间的角度，膜之间的相同距离 细菌视紫红质中的跨越螺旋），并用棕榈酰化的 在两端的残基。棕榈酰化残留物的使用将 促进肽与膜的结合。在这方面 我们设计了一种肽， 在完整的受体中发现。 将测试每种肽的结合和活化， G蛋白肽将在水溶液中进行构象检查， 溶液和在胶束存在下作为膜的模拟物 环境该项目的这一部分已经完成了两个 缩氨酸下一步是表征构象转变 肽与G蛋白结合后所经历的变化。通过 使用转移的NOE来改变小肽的构象， 与异源三聚体G蛋白（α、β和γ亚基）结合 将被确定;仅使用α亚基的实验已经被确定。 计划好了从细胞质环的构象特征， 受体，而自由和结合的G蛋白的重要 构成和结构特征的生物活性“将是 阐明。这种洞察力将使我们能够设计分子来调节 （增强或抑制）信号转导中的这一重要步骤。 我们目前正在使用上述方法来检查G- 蛋白偶联的甲状旁腺激素受体负责 维持钙水平和骨代谢。的表征 这种受体与其相关的G蛋白（G-s，和 G-q）将为我们提供另一个分子设计的目标， 帮助控制和调节钙水平。的信息 从这些研究中获得的信息将普遍适用于 G蛋白偶联受体的整个家族的表征。

We plan to characterize the association of G-proteins with their membrane bound receptors using experimental (including nuclear magnetic resonance, primarily transferred NOEs) and theoretical (including computer simulations for structure refinement and molecular modeling) techniques. The synthesis of peptides containing different cytoplasmic portions of the receptor has been partially realized and will be completed daring this granting period. The peptides will be examined in three different forms; linear- cyclized (utilizing a methylene linker to maintain a distance of 12 Angstroms between the termini, the same distance between the membrane spanning helices in bacteriorhodopsin) and cyclized with palmitoylated residues at both termini. The use of palmitoylated residues will facilitate the association of the peptide to the membrane. In this regard we have designed a peptide that should adopt a similar conformation to that found in the intact receptor. Each of the peptides will be tested for association and activation of the G-protein. The peptides will be conformationally examined both in aqueous solution and in the presence of micelles as a mimetic for a membrane environment. This portion of the project has been completed for two peptides. The next step is to characterize the conformational transition that the peptide undergoes upon association with the G-protein. Through the use of transferred NOEs the conformation of the small peptide while bound to the heterotrimeric G-protein (alpha, beta and gamma subunits) will be determined; experiments utilizing only the alpha-subunit have been planned. From the conformational features of the cytoplasmic loops of the receptor while free and bound to the G-protein the important constitutional and structural features far biological activity' will be elucidated. This insight will allow us to design molecules to regulate (enhance or inhibit) this important step in signal transduction. We are currently using the methods described above to examine the G- protein coupled parathyroid hormone receptor responsible for the maintenance of calcium levels and bone metabolism. The characterization of the interaction between this receptor and its related G-proteins (G-s, and G-q) will provide us with another target in the design of molecules to help in the control and regulation of calcium levels. The information obtained from these studies will be generally applicable for the characterization of the whole family of G-protein coupled receptors.