Conformational Dynamics of G Protein-Coupled Receptors at the Single-Molecule Level

项目总结/摘要 我的研究项目的总体目标是了解人类G蛋白偶联的动态行为 受体（GPCR）驱动GPCR复合物与药物和伴侣信号蛋白的组装， 单分子水平。GPCR是一种感觉膜蛋白，可以识别多种激素，药物， 和神经递质，代表了FDA批准的治疗药物靶向的最大一类蛋白质。的 GPCR的能量景观是复杂的，并且由具有不同功能的多种构象异构体填充， 结构.虽然一些GPCR构象异构体的结构已经通过X射线晶体学表征， 和冷冻EM，这些不同构象的寿命和它们的交换率大多是未知的。我们 旨在使用单分子荧光（SMF）绘制GPCR复合物的能量景观，这使得 实时和在重现细胞环境的环境中研究GPCR动态。在 从长远来看，我们的目标是应用这些信息，以提高我们对疾病相关突变如何 改变这些能量景观，这可能最终指导新疗法的设计。这项建议旨在 应用SMF绘制两个人类GPCR的能量景观。首先，我们将研究构象 图1示出了代表性A类人GPCR，A2 A腺苷受体（A2 AAR）的动力学。A2 AAR提供了一个 单分子荧光研究的重要基准，并将使我们能够比较我们的实验 从动态测量到计算预测。我们的研究将揭示两者的异同 在不同的A类GPCR之间的信号传导机制，也将首次显示如何脂质在 该双层膜可作为GPCR功能的变构调节剂。在第二个方向，我们将使用 SMF研究人胰高血糖素受体（GCGR）的构象动力学。GCGR是一种激素- 结合B类GPCR，其由中心代谢物之一胰高血糖素激活。GCGR对葡萄糖至关重要 是2型糖尿病治疗的有效药物靶点。GCGR的晶体和冷冻电镜结构 已经表明大的胞外结构域似乎与跨膜结构域协同作用， 激素和小分子，但配体结合的动力学还不清楚。我们的研究 GCGR将实时揭示细胞外结构域识别配体的机制， 功能相关的跨膜结构域的动态波动。这些研究将使我们能够比较 与激素和小分子形成复合物的动力学，以了解 配体识别中的胞外结构域。这将帮助我们了解不同化学物质的配体 结构和药理学功效影响受体活化途径，并将最终有助于 设计和筛选具有定制药理学反应的GPCR靶向药物。

PROJECT SUMMARY/ABSTRACT The overall goal of my research project is to understand how the dynamic behavior of human G protein-coupled receptors (GPCRs) drives the assembly of GPCR complexes with drugs and partner signaling proteins at a single-molecule level. GPCRs are sensory membrane proteins that recognize a wide array of hormones, drugs, and neurotransmitters, representing the largest class of proteins targeted by FDA-approved therapeutics. The energy landscape of GPCRs is complex and populated by multiple conformers with distinct functions and structures. While the structures of some GPCR conformers have been characterized by x-ray crystallography and cryo-EM, the lifetimes of these different conformations and their rates of exchange are mostly unknown. We aim to map the energy landscape of GPCR complexes using single-molecule fluorescence (SMF), which enables the investigation of GPCR dynamics in real-time and in environments that recapitulate the cellular milieu. In the long term, we aim to apply this information to improve our understanding of how disease-associated mutations alter these energy landscapes, which may ultimately guide the design of new therapeutics. This proposal aims to apply SMF to map the energy landscapes of two human GPCRs. First, we will investigate the conformational dynamics of a representative class A human GPCR, the A2A adenosine receptor (A2AAR). A2AAR provides an important benchmark for single-molecule fluorescence studies and will enable us to compare our experimental measurements of dynamics to computational predictions. Our studies will reveal both similarities and differences in mechanisms of signaling between different class A GPCRs and will also show for the first time how lipids in the bilayer membrane can act as allosteric modulators of GPCR function. In the second direction, we will use SMF to study the conformational dynamics of the human glucagon receptor (GCGR). GCGR is a hormone- binding class B GPCR that is activated by one of the central metabolites, glucagon. GCGR is critical to glucose homeostasis and is a validated drug target for type 2 diabetes therapy. Crystal and cryo-EM structures of GCGR have shown that the large extracellular domains appear to act in concert with the transmembrane domain to bind hormones and small molecules, but the dynamics of ligand binding are as yet not understood. Our studies of GCGR will reveal in real-time the mechanisms of ligand recognition by the extracellular domain and quantify function-related dynamic fluctuations of the transmembrane domains. These studies will allow us to compare the dynamics of complex formation with hormones and with small molecules to understand the role of the extracellular domain in ligand recognition. This will help us understand how ligands with different chemical structures and pharmacological efficacies affect the receptor activation pathways and will ultimately aid in the design and screening of GPCR-targeted drugs with tailored pharmacological responses.