GONADOTROPIN PHYSICAL CHEMISTRY

该提案的主要重点是确定三维 人绒毛膜促性腺激素及其受体的结构。 解决方案 亚基的结构和激素的活性形式 他们的受体界面将使激素激动剂的设计成为可能 可能有用的生育剂的拮抗剂。 虽然我们已经通过晶体学解析出了hCG的结构 技术，生物活性激素的结构尚未 已确定。 我们计划彻底解决native的结构 糖基化 hCG 以及 NMR 检测的亚基作为此部分的一部分 提案。 NMR 技术采用新颖的同位素标记方法 是在这个实验室开发的。 这种 NMR 标记是一项突破 结构研究。 它将能够确定三项 溶液中糖蛋白的空间结构。 此外，这些 NMR研究可以提供有关亚基/亚基的动态信息 传统 X 射线无法实现的相互作用 晶体学。 例如，绑定后循环的移动 受体可以在同位素标记的蛋白质中可视化。 第二个研究领域将采用创新的表达系统， “噬菌体展示”产生大量的细胞外结构域 LH/CG 受体。 胞外结构域将从融合物中制备 丝状噬菌体表面表达的蛋白质。 噬菌体 将在 13C、15N 标记的培养基中生长。 细胞外部分 显示与外壳蛋白融合将从噬菌体中释放 并纯化游离的和与 hCG 结合的 hCG，用于 NMR 结构研究。 这一应用对生物学具有重要意义，因为它将扩展我们的研究范围。 了解荷尔蒙的结构和功能 繁殖。 它将提供新的信息，使 设计类似物来控制其行为。 这些研究也将 为基础研究做出重大贡献，因为我们将开发和 在我们的实验过程中实施新技术。

The primary focus of this proposal is to determine the three-dimensional structure of human chorionic gonadotropin and its receptor. Solutions of the structures of the subunits and the active form of the hormone and their receptor interface will enable design of hormones agonists and antagonists that maybe useful fertility agents. Although we have solved the structure of hCG by crystallographic techniques, the structure of the biologically active hormone has not been determined. We plan to solve the structure of native fully glycosylated hCG as well as the subunits by NMR as part of this proposal. The NMR technique uses a novel isotopic labeling method developed in this laboratory. This NMR labeling is a breakthrough for structural studies. It will enable the determination of the three- dimensional structure of a glycoprotein in solution. In addition, these NMR studies can provide dynamic information about subunit/subunit interactions that is not available from conventional x-ray crystallography. For example, movement of loops upon binding to receptor can be visualized in isotopically labeled proteins. The second area of study will employ the innovative expression system, "phage display" to generate quantities of the extracellular domain of the LH/CG receptor. The extracellular domain will be prepared from a fusion protein expressed on the surface of a filamentous phage. The phage will be grown in 13C, 15N labeled media. The extracellular portion which is displayed fused to the coat protein will be liberated from the phage and purified both free and bound to hCG for NMR structural studies. This application is of significance to biology since it will expand our understanding of the structure and function of the hormones of reproduction. It will provide new information that will enable the design of analogues to control their actions. These studies will also contribute significantly to basic research in that we will develop and implement new technologies in the course of our experiments.