The molecular basis of alcohol's actions

描述（由申请人提供）：目标和健康相关问题。与暴露于乙醇相关的致醉作用与几种神经递质受体（配体门控离子通道）活性的变化有关。γ-氨基丁酸（GABA）和N-甲基-D-天冬氨酸（NMDA）受体急性暴露于乙醇直接涉及胎儿酒精综合征的发展，也在酒精中毒和酒精依赖的成人。越来越多的证据表明，乙醇结合到这些受体上的特定位点，并诱导构象变化，改变其活性。对酒精敏感蛋白中酒精结合位点的研究将为开发控制酒精中毒和酒精依赖的药物提供潜在的靶点。目前，由于研究膜蛋白的内在困难，这些重要的乙醇敏感蛋白的潜在结合位点的性质没有直接的结构信息。LUSH是一种来自果蝇的新型醇结合蛋白，可识别乙醇、正丙醇和正丁醇。我们最近解决了与乙醇的复合物中LUSH的结构。该提案的长期目标是详细描述该结合位点的分子性质，以确定酒精结合特异性的分子基础。结合到一系列醇的LUSH的结构将使用X射线晶体学方法来解决，以揭示非酶蛋白中醇特异性的分子图像。将使用生物物理和光谱方法分析不同醇对结合亲和力和蛋白质稳定性的影响，并且差异与溶液中蛋白质结构的变化相关。特定氨基酸在醇结合和蛋白质功能中的作用将使用定点诱变来测试，以改造具有修饰的配体结合特性的蛋白质。最终的目标是开发一个模型，在酒精敏感的蛋白质，这将有助于在酒精引起中毒和毒性的分子基础的理解特定的酒精结合位点。

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.