Probing Receptor Structures with Unnatural Amino Acids

描述(由申请人提供)：发展对突触交流的分子机制的理解，从而形成学习、记忆和感官感知的基础，是现代科学的中心挑战之一。在这项提案中，重点放在一个在这一过程中发挥核心作用的神经受体大家族，即配体门控离子通道(LGIC)。这些蛋白质介导快速突触传递，它们是阿尔茨海默病、帕金森氏病、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定和有效的药物-受体相互作用的确切化学相互作用，包括具有治疗价值的药物和滥用药物。这将对开发具有更高选择性和效价的新药具有相当大的价值。药物与LGIC的结合导致蛋白质结构的巨大变化，导致蛋白质内离子通道的打开，这对细胞来说是一个深刻的信号事件。这一“门控”过程是受体功能的核心，也将是研究的另一个重点。LGICs和其他神经受体是复杂的、多亚单位的、完整的膜蛋白。因此，结构生物学的强大工具--X射线结晶学和核磁共振光谱学--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到活细胞表达的蛋白质中。这种方法允许对受体进行几乎无限的修饰。可以进行微妙的更改以揭示键绑定交互。更戏剧性的变化引入了生物物理工具来探测结构和功能。当与电生理学的力量相结合时，非天然氨基酸突变成为一种强大的、广泛适用的工具，用于解开分子神经生物学中这些核心角色的关键特征。

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.