The molecular basis of alcohol's actions.

描述(由申请人提供)：目标和与健康有关的问题。与接触乙醇相关的醉人效应与几种神经递质受体的活性变化有关，这些受体是配体门控离子通道。酒精对γ-氨基丁酸(GABA)和N-甲基-D-天冬氨酸(NMDA)受体的急性暴露直接与胎儿酒精综合征的发生有关，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置结合，并诱导构象变化，从而改变它们的活性。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的靶点。目前还没有关于这些重要的乙醇敏感蛋白潜在结合位点的性质的直接结构信息，因为在研究完整的膜蛋白方面存在固有的困难。LUSH是一种来自果蝇的新型酒精结合蛋白，可以识别乙醇、正丙醇和正丁醇。我们最近已经解决了乙醇络合物中的LUSH结构。这项建议的长期目标是详细描述这个结合位点的分子性质，以便确定酒精结合特异性的分子基础。我们将用X射线结晶学的方法解开与一系列醇结合的LUSH的结构，以揭示非酶蛋白质中酒精专一性的分子图像。不同的醇对结合亲和力和蛋白质稳定性的影响将通过生物物理和光谱方法进行分析，这种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定点突变来测试，以设计具有修改的配体结合特性的蛋白质。最终目标是为酒精敏感蛋白中特定的酒精结合部位建立一个模型，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.