Properties of Specific Alcohol Binding Sites

特定醇结合位点的特性

• 批准号: 6545044
• 负责人: JAMES Robert TRUDELL
• 金额: $ 23.55万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6545044
• 关键词: GABA receptor; acetylcholine; alcohols; binding sites; chemical models; cysteine; disulfide bond; glycine receptors; membrane channels; model design /development; molecular biology information system; molecular dynamics; nicotinic receptors; point mutation; receptor binding; serotonin receptor

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism are major health problems. It is likely that a solution to these problems will require an understanding of the effects of alcohol on specific ion channels and the kinases that modulate them. Specific binding sites for alcohols have recently been described in the transmembrane domain of the superfamily of glycine, GABA, nicotinic acetylcholine, and 5-HT3 receptors. Our hypothesis is that alcohols bind within cavities that are bounded by transmembrane segments of these receptors. Our goal is to define the properties of those sites that regulate binding and efficacy of alcohols. These binding sites may provide a common motif for binding of alcohols within other classes of ion channels. We will build computational models of binding sites and design specific site-directed mutations to test this hypothesis. These mutations will be expressed and tested by our collaborators, Drs. R. Adron Harris and S. John Mihic, in separately funded experiments. Specifically: Aim 1. We will define specific amino acid residues that determine the "cutoff" length of long-chain alcohols. We have previously shown that mutation of S267 in transmembrane segment 2 (TM2) and A288 in TM3 of the glycine alphal receptor can change the alcohol "cutoff' from heptanol to dodecanol. We will develop computational molecular models that allow us to suggest mutations that will determine additional residues in TM1 and TM4 that may also form "walls" of the putative binding cavities. We will refine our models by iterations in which we optimize the structure of an initial model, use it to predict mutations, test if the model is consistent with the resulting experimental data, and then modify the model in a way that would better fit the data. Aim 2. We will determine the structural requirements of alcohols for potentiation of agonist potency by providing models in which a series of alcohol analogs are covalently linked to site-directed cysteine mutations in the putative binding cavities. Since we will know that a single alcohol analog is bound to the putative site, we can distinguish binding from efficacy. Aim 3. We will define the proximity of amino acid residues important for alcohol potentiation of agonists by building models that predict double site-directed cysteine mutations that are appropriate for cross-linking. We will predict pairs of residues that could be linked by direct disulfide formation or with bi-functional methanethiosulfonate reagents with 1-5 carbon spacers. In summary, these computational studies will provide new knowledge about determinants of alcohol binding and efficacy.

描述（由申请人提供）：酗酒和酒精中毒是主要的健康问题。解决这些问题的解决方案很可能需要了解酒精对特定离子通道和调节它们的激酶的影响。最近在甘氨酸，GABA，烟碱乙酰胆碱和5-HT3受体的超家族的跨膜结构域中描述了醇的特定结合位点。我们的假设是，醇在这些受体的跨膜段界定的空腔内结合。我们的目标是定义调节酒精结合和功效的那些位点的特性。这些结合位点可能会为醇在其他类别的离子通道内的结合提供一个共同的基序。我们将构建结合位点的计算模型和设计特定位置定向的突变以检验该假设。这些突变将由我们的合作者Drs表示和测试。 R. Adron Harris和S. John Mihic，在分别资助的实验中。具体来说：AIM 1。我们将定义确定长链醇长度“截止”长度的特定氨基酸残基。我们先前已经表明，在跨膜段2（TM2）中S267的突变和在甘氨酸α受体TM3中的A288突变可以改变酒精“从己醇到十二烷醇”。我们将开发计算分子模型，从而使我们能够在TM1和TM4中构成tm4 wals'''''''''''''''cutionational Mocy of''''cutionational Mocyally'''crout'''''cutectitational Mocely''croun'''''通过优化初始模型的结构，使用它来预测突变，测试模型是否与所得的实验数据一致，然后以更好地符合数据的方式来修改模型我们会知道，单个酒精类似物与推定位点结合，我们可以将结合与功效区分开。 AIM 3。我们将通过构建预测适合交叉链接的双位定向半胱氨酸突变的模型来定义对激动剂的葡萄酒增强氨基酸残基的接近性。我们将预测可能通过直接二硫键形成或具有1-5个碳垫片的双官能甲甲基磺酸盐试剂对残留物。总而言之，这些计算研究将提供有关酒精结合和功效决定因素的新知识。