Characterizing the Dynamic Response of the Estrogen Receptor to Agonists and Antagonists by Multi-frequency Electron Spin Resonance Spin- Labeling

摘要

The overall objective of this project is to characterize the detailed structural and dynamic response of the estrogen receptor ligand binding domain (ER-LBD) to a variety of ligands ranging from strong estrogens to strong antiestrogens using electron spin labeling. The technical aims for the initial period involved developing site-directed spin-labeled mutants of the ER-LBD and synthesizing new spin-labeled ligands for the proposed studies. We have optimized cell culture production of ER-LBD and expressed a number of single and doubly labeled mutants. In addition we have synthesized a new spin label for attachment to site-selected cysteine residues in this protein and made substantial progress towards the synthesis of spin-labeled ligands with a range of agonist/antagonist activities. Initial electron spin resonance studies have established that the selected label locations will be acceptably sensitive to the type of ligand present.