Anesthetic Binding Sites on Three-Helix Bundle Proteins

这项拟议的研究有一个长期目标，即从结构和动态的角度，在分子水平上了解吸入全身麻醉剂与蛋白质的相互作用。这将使用本实验室开发的检测麻醉剂与蛋白质靶标结合的方法来实现。主要的实验重点将是合成三α-螺旋束，这种束可以通过现有的技术在结构上进行修改。这些人工合成的蛋白质作为跨膜α-螺旋束的简化模型，是中枢神经系统中普遍存在的离子通道和神经递质受体的结构成分，是目前全身麻醉药的首选靶点。目前对膜蛋白的结构理解排除了它们用于精确检查麻醉剂-蛋白质络合的可能性。然而，建议使用简化的、定义良好的天然蛋白质跨膜结构域模型，有助于使用各种光谱方法和X射线结晶学直接确定麻醉剂结合部位的结构特征。这将提供一个详细的框架来评估疏水性、极性和蛋白空腔对麻醉剂结合的贡献，从而深入了解特定分子相互作用对麻醉剂络合的相对重要性。这一信息将为挥发性全身麻醉药的体内结合部位的结构组成提供指南。麻醉剂与蛋白质靶标结合的后果将使用荧光各向异性和蛋白质热力学稳定性等蛋白质动力学指标来确定，目的是加深我们对结合麻醉剂如何改变蛋白质功能的理解。拟议的研究基于已报道的氟烷与二聚体四α-螺旋束结合的结果，以(I)确定麻醉剂结合部位的结构，以及(Ii)获得与结合麻醉剂结合的蛋白质的原子级X射线晶体结构。最终，这些模型系统的使用将提供关于这些重要的临床化合物如何在分子水平上与中枢神经系统中的潜在靶点相互作用的基本信息，并将建立一个测试这种关联的框架，与天然膜蛋白。对该模型系统上麻醉剂结合位点的精确结构描述将允许在蛋白质数据库中重点搜索现有条目和未来条目上的潜在结合位点。

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.