Probing Receptor Structures with Unnatural Amino Acids

用非天然氨基酸探测受体结构

• 批准号: 7108538
• 负责人: DENNIS A DOUGHERTY
• 金额: $ 34.35万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7108538
• 关键词: NMDA receptors; Xenopus oocyte; acetylcholine; aminoacid analog; binding sites; conformation; inhibitor /antagonist; ligands; neural transmission; nicotinic receptors; nuclear magnetic resonance spectroscopy; protein structure function; receptor binding; receptor expression; serotonin receptor; structural biology; tissue /cell culture; voltage /patch clamp

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.

描述（由申请人提供）：了解突触通讯的分子机制，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。在该提案中，重点是在这一过程中发挥核心作用的一大类神经受体，即配体门控离子通道（LGIC）。这些蛋白质介导快速突触传递，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治疗方法的目标。这项工作的一个主要目标是加深对精确化学相互作用的理解，这些化学相互作用会导致特定且有效的药物-受体相互作用，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 的结合会引起蛋白质的巨大结构变化，从而导致蛋白质内离子通道的打开，这对细胞来说是一个意义深远的信号事件。这种“门控”过程对于受体的功能至关重要，这将是研究的另一个焦点。 LGIC 和其他神经受体是复杂的多亚基完整膜蛋白。因此，结构生物学的强大工具——X射线晶体学和核磁共振波谱——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。可以进行细微的改变来揭示关键的绑定交互。更显着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，非天然氨基酸诱变成为一种强大的、广泛适用的工具，用于揭示分子神经生物学中这些核心参与者的关键特征。