G PROTEIN/RECEPTOR ASSOC--STRUCTURAL CHARACTERIZATION

我们计划表征G蛋白与它们的膜的联系 结合受体使用实验(包括核磁共振， 主要是转移的NOES)和理论(包括计算机 用于结构优化和分子建模的模拟)技术。 含不同胞质部分的多肽的合成 受体已经部分实现，并将在此基础上完成 授权期。这些多肽将以三种不同的形式进行检测； 线性环化(利用亚甲基连接物保持距离 末端之间12埃，膜之间的距离相同 细菌视紫红质中的螺旋)和棕榈酰化的环化 两个终点站的残留物。棕榈酰化残留物的使用将 促进多肽与膜的结合。在这方面， 我们已经设计了一种多肽，它应该具有类似的构象 这是在完整的受体中发现的。 将测试每一种多肽的结合和激活 G蛋白。这些多肽将在水溶液中进行构象研究 溶液和在胶束存在的情况下作为膜的模拟物 环境。这部分工程已经完成两年了。 多肽。下一步是描述构象转变的特征 多肽与G蛋白发生关联。穿过 利用转移NOES改变小肽的构象 与异源三聚体G蛋白(α、β和伽马亚基)结合 将被确定；仅使用阿尔法亚单位的实验已经被确定 有计划的。根据胞质环的构象特征 受体在游离和结合G蛋白中的重要作用 生物活动的构成和结构特征将是 已澄清。这种洞察力将使我们能够设计分子来调节 (加强或抑制)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- 蛋白偶联甲状旁腺激素受体参与 维持钙水平和骨骼代谢。人物形象的塑造 该受体与其相关的G蛋白(G-S和G-G-R)之间的相互作用 G-Q)将为我们在分子设计方面提供另一个目标 有助于控制和调节钙水平。这些信息 从这些研究中得出的结论将普遍适用于 整个G蛋白偶联受体家族的特性。

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.