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Development and Applications of Bioorthogonal Chemistry: Administrative Supplement for Equipment

生物正交化学的发展与应用：设备管理补充

基本信息

批准号：
10581256
负责人：
Qing Lin
金额：
$ 7.44万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2023-12-31
项目状态：
已结题

项目摘要

Development and Applications of Bioorthogonal Chemistry: Administrative Supplement for Equipment ABSTRACT We have a long-standing interest in developing reactivity-based chemical tools to address significant biological problems that are difficult to solve using conventional molecular biology techniques. In our MIRA application, we plan to continue our studies of orthogonal chemical reactivity at the chemistry-biology interface and pursue the following two related projects. In Project 1, we will construct the FRET-based biosensors of GLP-1R and GCGR using bioorthogonal chemistry techniques. These biosensors will then be employed to probe the conformational dynamics during the ligand-induced receptor activation and signaling in live cells. A new set of fluorescence 'turn-on' reagents will be designed for bioorthogonal labeling of the intracellular loop 3 of GLP-1R and GCGR to allow single-cell intra- and inter-molecular FRET analysis of receptor dynamics in live cells. In Project 2, we will develop a genetically encoded chemical crosslinker containing an alkyne group and apply this chemical crosslinker to map the time-dependent GLP-1R and -arrestins interactomes by mass spectrometry in response to ligand stimulation. We expect these studies to validate new bioorthogonal tools for real-time monitoring of protein conformation and protein-protein interactions in live cells. Also, we will gain novel insights into the GLP-1R and GCGR activation dynamics and structural basis of biased signaling that are crucial for the development of targeted therapies for the treatment of diabetes and obesity. This Administrative Supplement requests the acquisition of an Agilent 1260 Infinity II LC System with Diode Array Detector. This system will help us characterize the bioorthogonal reagents' reaction kinetics and selectivity at higher throughput and greater accuracy.

生物正交化学的发展与应用：设备管理补充抽象的我们长期以来对开发基于反应性的化学工具来解决重大的生物问题有着浓厚的兴趣。使用传统分子生物学技术难以解决的问题。在我们的 MIRA 应用程序中，我们计划继续研究化学-生物学界面的正交化学反应性并追求以下两个相关项目。在项目1中，我们将构建基于FRET的GLP-1R和 GCGR 使用生物正交化学技术。这些生物传感器将被用来探测活细胞中配体诱导的受体激活和信号传导过程中的构象动力学。一套新的荧光“开启”试剂将被设计用于 GLP-1R 细胞内环 3 的生物正交标记 GCGR 可对活细胞中的受体动态进行单细胞内和分子间 FRET 分析。在项目2，我们将开发一种含有炔基的基因编码化学交联剂并应用这种化学交联剂可按质量绘制时间依赖性 GLP-1R 和 -arrestins 相互作用组响应配体刺激的光谱测定。我们期望这些研究能够验证新的生物正交工具实时监测活细胞中的蛋白质构象和蛋白质-蛋白质相互作用。同时，我们还将收获对 GLP-1R 和 GCGR 激活动力学以及偏向信号传导的结构基础的新见解对于开发治疗糖尿病和肥胖症的靶向疗法至关重要。这行政补充要求购买配备二极管的 Agilent 1260 Infinity II 液相色谱系统阵列探测器。该系统将帮助我们表征生物正交试剂的反应动力学和具有更高通量和更高准确度的选择性。