Decoding Structural Determinants of Efficacy and Specificity in a GPCR Subfamily

项目摘要 G蛋白偶联受体(GPCRs)是最大的膜蛋白家族，也是一大类药物 靶点，通过经历构象变化将细胞外刺激转化为细胞内反应 使它们能够结合和激活信号蛋白。找出激活不足的机制-- 从配体结合口袋传播的原子级重排，在分子上诱导大规模运动 细胞内表面--可以极大地影响药物发现，但需要测量构象变化 在多种时间和空间尺度上。因此，只有少数GPCR的激活机制具有 在原子水平上被仔细绘制，阻碍了高选择性和高选择性治疗药物的合理设计 几乎没有副作用。为那些在治疗上有希望的GPCR揭示这些机制 多聚体具有较大的胞外结构域，需要能够连接多个尺度的生物物理方法。 在我的博士生涯中，我拥有使用基于物理的模拟来捕捉构象变化的专业知识 膜蛋白具有很高的空间和时间分辨率，但分子动力学的计算代价 (MD)限制了可获得的时间尺度和对蛋白质家族许多成员的调查。作为博士后研究员 在加州大学伯克利分校，我一直在进行实验研究，以填补与经典MD模拟相关的空白。 我学会了在监督下进行氢-重质谱(HDX-MS) 苏珊·马尔库塞博士的研究，使我能够量化蛋白质中结构元素的局部稳定性。更多 最近，我推行了另一种策略，单分子Förster共振能量转移(SmFRET)， 为了量化蛋白质构象景观中的相对状态种群，在Dr。 埃胡德·伊萨科夫。我将继续对一类难以用药的GPCRs进行计算和实验研究 代谢性谷氨酸受体(MGluRs)MGluR有一个复杂的拓扑结构：配体结合域 这些GPCR中的(LBD)将配体的作用横向地传递到邻近的亚基，并在细胞内传递到 跨膜结构域。在目标1中，我将确定配体结合影响的机制 在单个亚基内的构象变化；穿过二聚体界面；以及在下面，到跨膜 域。在目标2中，我将研究mGluR家族中的序列变异如何导致惊人的广泛 先前观察到的八种mGluR亚型的谷氨酸亲和力和有效性的范围。在《目标3》中，我会 研究内源性细胞外结合伙伴如何调节mGluRs以影响下游激活。 这项工作将为研究多结构域的构象变化机制提供一个通用的策略 蛋白质，并将使变构配体的发现，可以选择性地靶向特定的受体，而 引出特定的信令输出。加州大学伯克利分校丰富的科学环境，以及我的支持 杰出的导师，将使我能够在为学术生涯做准备的同时接受实验生物物理学方面的培训。

Project Summary G protein–coupled receptors (GPCRs), the largest family of membrane proteins and a major class of drug targets, convert extracellular stimuli into intracellular responses by undergoing conformational changes that enable them to bind and activate signaling proteins. Identifying the mechanisms that underly activation––the atomic-level rearrangements that propagate from the ligand-binding pocket to induce large-scale motions on the intracellular surface––can powerfully impact drug discovery but requires measurement of conformational change at multiple temporal and spatial scales. Thus, activation mechanisms for only a small number of GPCRs have been carefully mapped at the atomic level, hindering the rational design of therapeutics with high selectivity and few side effects. Uncovering these mechanisms for those therapeutically promising GPCRs that operate as multimers, with large extracellular domains, requires biophysical approaches that can bridge multiple scales. From my Ph.D., I have expertise in using physics-based simulations to capture conformational change in membrane proteins with high spatial and temporal resolution, but the computational cost of molecular dynamics (MD) limits accessible timescales and investigation of many members of a protein family. As a postdoctoral fellow at UC-Berkeley, I have pursued experimental studies to fill in the gaps associated with classical MD simulations. I have learned to carry out hydrogen-deuterium exchange–mass spectrometry (HDX-MS) under the supervision of Dr. Susan Marqusee, enabling me to quantify the local stability of structural elements in a protein. More recently, I have pursued an additional strategy, single-molecule Förster Resonance Energy Transfer (smFRET), to quantify the relative populations of states in a protein conformational landscape, under the guidance of Dr. Ehud Isacoff. I will pursue computational and experimental studies of a difficult-to-drug class of GPCRs, the metabotropic glutamate receptors (mGluRs). The mGluRs have a complex topology: the ligand-binding domains (LBD) of these GPCRs transmit a ligand's effects laterally, to the neighboring subunit, and intracellularly, to the transmembrane domain. In Aim 1, I will determine the mechanisms by which ligand binding affects conformational changes within a single subunit; across the dimer interface; and below, to the transmembrane domain. In Aim 2, I will investigate how sequence variation in the mGluR family leads to the strikingly broad range of glutamate affinity and efficacy previously observed for the eight mGluR subtypes. In Aim 3, I will investigate how endogenous extracellular binding partners modulate mGluRs to affect downstream activation. This work will provide a general strategy for investigating mechanisms of conformational change for multi-domain proteins and will enable discovery of allosteric ligands that can selectively target a particular receptor while eliciting a specific signaling output. The rich scientific environment of UC-Berkeley, along with the support of my outstanding mentors, will enable me to train in experimental biophysics while preparing for an academic career.