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结构 解决方案 亚单位的结构和激素的活性形式 并且它们的受体界面将使得能够设计激素激动剂， 拮抗剂，可能有用的生育剂。 虽然我们已经通过晶体学方法解决了hCG的结构， 技术，生物活性激素的结构没有 确定了 我们计划完全解决原生的结构 糖基化的hCG以及通过NMR作为其一部分的亚基 提议 核磁共振技术使用一种新的同位素标记方法 在这个实验室里开发的。 这种核磁共振标记是一个突破， 结构研究。 它将能够确定三个- 溶液中糖蛋白的三维结构。 另外这些 NMR研究可以提供关于亚基/亚基的动态信息 传统X射线无法提供的相互作用 结晶学 例如，在绑定到 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”以产生大量的噬菌体的胞外结构域。 LH/CG受体。 细胞外结构域将通过融合制备 在丝状噬菌体表面表达的蛋白质。 噬菌体 将在13 C、15 N标记的培养基中生长。 细胞外部分， 与外壳蛋白融合的噬菌体将从噬菌体中释放出来 并纯化游离的和结合hCG的用于NMR结构研究。 这一应用对生物学具有重要意义，因为它将扩大我们的 了解激素的结构和功能， 生殖 它将提供新的信息， 设计类似物来控制它们的行为。 这些研究还将 为基础研究做出重大贡献，我们将开发和 在我们的实验过程中应用新技术。

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.