GONADOTROPIN PHYSICAL CHEMISTRY

本方案的主要重点是确定立体

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.