Anesthetic Binding Sites on Three-Helix Bundle Proteins

三螺旋束蛋白上的麻醉结合位点

• 批准号: 6712086
• 负责人: JONAS S JOHANSSON
• 金额: $ 26.63万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6712086
• 关键词: X ray crystallography; alkanes; anesthetics; binding sites; bioenergetics; chemical binding; chloroform; circular dichroism; conformation; ethers; fluorescence spectrometry; gas chromatography; halothane; inhalation anesthesia; membrane proteins; nuclear magnetic resonance spectroscopy; protein purification; protein structure function; synthetic protein; thermostability

The proposed research has the long-range goal of providing an understanding of how the inhaled general anesthetics interact with proteins, from structural and dynamic points of view, at the molecular level. This will be achieved using approaches developed in this laboratory to detect anesthetic binding to protein targets. The primary experimental focus will be synthetic three-alpha-helix bundles that can be structurally modified by established techniques. These synthetic proteins serve as simplified models for the bundles of transmembrane alpha-helices that are ubiquitous structural components of ion channels and neurotransmitter receptors in the central nervous system, and are the favored targets for general anesthetics at present. The current structural understanding of membrane proteins precludes their use to precisely examine anesthetic- protein complexation. However, the proposed use of simplified, well-defined, models of the transmembrane domains of native proteins lend themselves to the direct determination of the structural features of anesthetic binding sites using various spectroscopic approaches and X-ray crystallography. This will provide a detailed frame to evaluate hydrophobic, polar, and protein cavity contributions to anesthetic binding, providing insight into the relative importance of specific molecular interactions for anesthetic complexation. This information will provide guidelines for the structural composition of in vivo binding sites for volatile general anesthetics. The consequences of anesthetic binding to protein targets will be determined using measures of protein dynamics such as fluorescence anisotropy and protein thermodynamic stability, with the goal of furthering our understanding of how a bound anesthetic might alter protein function. The proposed studies build on the reported findings on halothane binding to dimeric four-alpha-helix bundles to (i) define the structure of the anesthetic binding site, and (ii) obtain atomic-level X-ray crystal structures of protein with bound anesthetic. Ultimately, the use of such model systems will provide fundamental information concerning how these important clinical compounds interact with potential target sites in the central nervous system at the molecular level, and will establish a framework for testing such associations, with natural membrane proteins. A precise structural description of the anesthetic binding site on this model system will allow a focused search of the Protein Data Bank for potential binding sites on existing- and future entries.

拟议的研究具有远程目标，即从分子水平上从结构和动态观点来理解吸入的一般麻醉与蛋白质如何与蛋白质相互作用。 这将使用该实验室中开发的方法来检测与蛋白质靶标的麻醉结合。 主要的实验焦点将是合成的三α-螺旋束，可以通过既定技术在结构上修改。 这些合成蛋白是简化的模型，用于跨膜α-螺旋束，它们是中枢神经系统中离子通道和神经递质受体无处不在的结构成分，目前是全身麻醉的靶标。 当前对膜蛋白的结构理解无法精确检查麻醉蛋白的络合。 然而，建议使用各种光谱方法和X射线晶体学的简化，定义明确的模型的跨膜结构域的模型直接确定麻醉结合位点的结构特征。 这将提供一个详细的框架，以评估疏水性，极性和蛋白质腔对麻醉结合的贡献，从而洞悉特定分子相互作用对麻醉络合的相对重要性。 该信息将为挥发性通用麻醉剂的体内结合位点的结构组成提供指南。 麻醉结合与蛋白质靶标的后果将使用蛋白质动力学（例如荧光各向异性和蛋白质热力学稳定性）确定，目的是进一步了解我们对结合麻醉如何改变蛋白质功能的理解。 拟议的研究基于报道的关于硫烷与二聚体四螺旋螺旋束的发现，以（i）定义麻醉结合位点的结构，并且（ii）获得与结合麻醉的蛋白质的原子级X射线晶体结构。 最终，这种模型系统的使用将提供有关这些重要的临床化合物如何与分子水平中枢神经系统中潜在目标部位相互作用的基本信息，并将建立一个与天然膜蛋白一起测试此类关联的框架。 该模型系统上麻醉结合位点的精确结构描述将允许对蛋白质数据库进行重点搜索，以了解现有条目和将来条目的潜在结合位点。