Engineering a Transmitter Binding Site

描述(申请人提供)：生物医学的一个基本原理是小分子可以与特定的受体结合以触发生理反应。我们试图了解发生在神经肌肉乙酰胆碱受体的神经递质结合部位的分子事件的能量性质，当这一通道在非传导构象和离子传导构象之间‘门’时。这些知识将帮助我们理解配体-蛋白质复合体的生物物理机制，并将提高我们设计针对尼古丁(和其他)受体的新药的能力。基于结构的药物发现应该包含这样一个事实，即药物的作用依赖于配体与受体的非活性构象和活性构象的不同结合。为了解决这一问题，我们将使用单通道电生理学和动力学分析来研究未连接门控，这将使我们能够测量所有显著的激活平衡常数，并确定两个递质结合位点上侧链的能量贡献。我们还将估计小配体探针在野生型和突变型受体中的差异结合能。最终，这一知识将被用来设计对任意配体做出可预测反应的乙酰胆碱受体。公共卫生相关性：该项目中概述的实验为设计一种蛋白质以可预测的方式对特定药物做出反应确立了基本原则。这些原理可以应用于新药物的开发，以及理解药物导致蛋白质改变形状的机制。

DESCRIPTION (provided by applicant): A fundamental principle of biomedicine is that small molecules can bind to specific receptors to trigger physiological responses. We seek to understand the energetic nature of the molecular events that occur at the neurotransmitter binding sites of the neuromuscular acetylcholine receptor when this channel 'gates' between non-conducting and ion-conducting conformations. This knowledge will help us understand the biophysical mechanisms of ligand-protein complexes, and will advance our ability to design new drugs for nicotinic (and other) receptors. Structure-based drug discovery should incorporate the fact that drug action depends on the differential binding of ligands to inactive vs. active conformations of a receptor. To address this point we will use single-channel electrophysiology and kinetic analysis to study unliganded gating, which will allow us to measure all of the salient activation equilibrium constants and to ascertain the energetic contributions of the side chains at each of the two transmitter binding sites. We will also estimate differential binding energies for small ligand probes, in both wild type and mutant receptors. Eventually, this knowledge will be used to engineer acetylcholine receptors that respond predictably to arbitrary ligands. PUBLIC HEALTH RELEVANCE: The experiments outlined in this project establish the fundamental principles for engineering a protein to respond in predictable ways to specific drugs. These principles can be applied to the development of new pharmaceuticals, and to understanding the mechanism by which a drug causes a protein to change shape.