Probing Receptor Structures with Unnatural Amino Acids

描述（由申请人提供）：发展对突触通讯基础的分子机制的理解，从而形成学习，记忆和感官知觉的基础，是现代科学的核心挑战之一。在这个提议中，重点是在这个过程中发挥核心作用的一个神经受体大家族，配体门控离子通道（LGIC）。这些蛋白质介导快速突触传递，并且它们是阿尔茨海默病、帕金森病、精神分裂症、中风、学习和注意力缺陷以及药物成瘾的治疗方法的靶点。这项工作的一个主要目标是了解导致特异性和强效药物-受体相互作用的精确化学相互作用，包括具有治疗价值的药物和滥用药物。这对于开发具有改进的选择性和效力的新药物具有相当大的价值。药物与LGIC的结合诱导蛋白质中的大的结构变化，这导致蛋白质内的离子通道打开，这是细胞的深刻信号事件。这种“门控”过程是受体功能的核心，也将是另一个研究重点。LGIC和其他神经受体是复杂的、多亚基的、完整的膜蛋白。因此，结构生物学的强大工具-X射线晶体学和NMR光谱学-并不容易应用。这项工作利用体内无义抑制方法将非天然氨基酸掺入活细胞中表达的蛋白质中。这种方法允许几乎无限的受体修饰。可以进行细微的改变以揭示关键的结合相互作用。更引人注目的变化是引入了生物物理学工具来探测结构和功能。当与电生理学的力量相结合时，非天然氨基酸诱变成为一种强大的，广泛适用的工具，用于解开这些分子神经生物学中核心参与者的关键特征。

DESCRIPTION (provided by applicant): Developing an understanding of the molecular mechanisms that underlie synaptic communication, and hence form the basis of learning, memory, and sensory perception, stands as one of the central challenges of modern science. In this proposal, the focus is on one large family of neuroreceptors that plays a central role in this process, the ligand-gated ion channels (LGIC). These proteins mediate fast synaptic transmission, and they are the targets of therapeutic approaches to Alzheimer's disease, Parkinson's disease, schizophrenia, stroke, learning and attention deficits, and drug addiction. A major goal of this work is to develop an understanding of the precise chemical interactions that lead to specific and potent drug-receptor interactions, including both drugs of therapeutic value and drugs of abuse. This will be of considerable value in developing new pharmaceuticals with improved selectivities and potencies. Binding of a drug to an LGIC induces a large structural change in the protein that leads to the opening of an ion channel within the protein, a profound signaling event for the cell. This "gating" process is central to the function of the receptors, and it will be another focus of investigation. LGICs and other neuroreceptors are complex, multisubunit, integral membrane proteins. As such, the powerful tools of structural biology - x-ray crystallography and NMR spectroscopy - are not readily applicable. This work makes use of the in vivo nonsense suppression method for unnatural amino acid incorporation into proteins expressed in living cells. This approach allows almost limitless modification of the receptors. Subtle changes can be made to reveal key binding interactions. More dramatic changes introduce biophysical tools to probe structure and function. When combined with the power of electrophysiology, unnatural amino acid mutagenesis becomes a powerful, broadly applicable tool for unraveling the critical features of these central players in molecular neurobiology.