The molecular basis of alcohol's actions.

描述（由申请人提供）：目标和健康相关问题。与暴露于乙醇有关的中毒效应与几种配体门控离子通道的神经递质受体活动的变化有关。γ -氨基丁酸（GABA）和n -甲基d -天冬氨酸（NMDA）受体对乙醇的急性暴露与胎儿酒精综合征的发展直接相关，也与成人的酒精毒性和酒精依赖有关。越来越多的证据表明，乙醇与这些受体上的特定位点结合，引起构象变化，从而改变其活性。乙醇敏感蛋白中酒精结合位点的表征将为开发控制酒精中毒和酒精依赖的药物提供潜在靶点。由于研究整体膜蛋白固有的困难，目前还没有关于这些重要的乙醇敏感蛋白的潜在结合位点性质的直接结构信息。LUSH是一种来自果蝇的新型酒精结合蛋白，可识别乙醇、正丙醇和正丁醇。我们最近解决了与乙醇配合物中LUSH的结构。本提案的长期目标是详细表征该结合位点的分子性质，以确定醇结合特异性的分子基础。LUSH与一系列醇结合的结构将用x射线晶体学方法解决，以揭示非酶蛋白中醇特异性的分子图谱。采用生物物理和光谱学方法分析不同醇对结合亲和力和蛋白质稳定性的影响，并分析其差异与溶液中蛋白质结构的变化有关。特定氨基酸在酒精结合和蛋白质功能中的作用将通过位点定向诱变来测试，以修饰配体结合特性的工程蛋白质。最终目标是建立一个酒精敏感蛋白中特定酒精结合位点的模型，这将有助于理解酒精引起中毒和毒性的分子基础。

DESCRIPTION (provided by applicant): Objectives and Health Related Issues. The intoxicating effects associated with exposure to ethanol have been linked to changes in the activities of several neurotransmitter receptors that are ligand-gated ion-channels. Acute exposure of the gamma-amino-butyric acid (GABA) and N-methyl D-aspartate (NMDA) receptors to ethanol is directly implicated in the development of fetal alcohol syndrome and also in alcohol toxicity and alcohol dependency in the adult. There is increasing evidence that ethanol binds to specific sites on these receptors and induces a conformational change that modifies their activity. Characterization of alcohol-binding sites in ethanol sensitive proteins would provide potential targets for the development of pharmacological agents to control alcohol intoxication and alcohol dependency. At present there is no direct structural information available about the nature of potential binding sites of these important ethanol-sensitive proteins because of the inherent difficulties in studying integral membrane proteins. LUSH is a novel alcohol-binding protein from fruit flies that recognizes ethanol, n-propanol and n-butanol. We have recently solved the structure of LUSH in the complex with ethanol. The long-term goal of this proposal is to characterize the molecular nature of this binding site in detail in order to define the molecular basis for alcohol-binding specificity. The structure of LUSH bound to a series of alcohols will be solved using X-ray crystallographic methods to reveal a molecular picture of alcohol specificity in a nonenzymatic protein. The effect of different alcohols on binding affinity and protein stability will be analyzed using biophysical and spectroscopic methods, and the differences correlated with changes to the protein structure in solution. The role of specific amino acids in alcohol binding and protein function will be tested using site directed mutagenesis to engineer proteins with modified ligand-binding properties. The ultimate goal is to develop a model for specific alcohol-binding sites in alcohol-sensitive proteins that will aid in an understanding of the molecular basis of alcohols actions in causing intoxication and toxicity.