GONADOTROPIN PHYSICAL CHEMISTRY

这项建议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 它们的受体界面将使荷尔蒙激动剂和 可能有用的生育药剂的拮抗剂。 虽然我们已经用结晶学方法解决了hCG的结构问题 技术，生物活性激素的结构还没有 已经确定了。我们计划全面解决原生的结构 作为这一研究的一部分，核磁共振显示糖基化的hCG及其亚基 求婚。核磁共振技术使用了一种新的同位素标记方法 在这个实验室里研发的。这种核磁共振标记是对 结构研究。它将使我们能够确定以下三个方面- 糖蛋白在溶液中的空间结构。此外，这些 核磁共振研究可以提供有关亚基/亚基的动态信息 常规X射线所不具备的相互作用 结晶学。例如，在绑定到时循环的移动 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”，以产生大量的胞外区 黄体生成素/卵泡刺激素受体胞外结构域将从融合中制备 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。它的胞外部分 展示的是与外壳蛋白融合的噬菌体将被释放出来 并对游离和结合的hCG进行纯化，用于核磁共振结构研究。 这一应用对生物学具有重要意义，因为它将扩大我们的 对荷尔蒙结构和功能的理解 繁殖。它将提供新的信息，使 设计类似物来控制他们的行为。这些研究还将 对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.