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532 nm (green) laser

Company: Crystal Lasers
Catalog#: CL532-050-S
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Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
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2017-06-20
[Abstract]  Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and ... [摘要]  通过激动剂配体激活G蛋白偶联受体(GPCR)是通过从无活性受体构象向活性受体构象的转变来介导的。我们描述了一种新颖的单分子测定法,可以实时监测单个GPCR分子中的激活连锁构象转换。受体在跨膜螺旋6末端用Cy3荧光探针进行位点特异性标记,并在连接到显微镜载玻片的磷脂纳米圆盘中重构。然后通过单分子全内反射荧光显微镜随时间监测单个受体分子,显示无活性和活性样构象之间的自发转变。该测定提供关于无活性和活性受体构象的平衡分布以及构象交换的速率常数的信息。实验可以在不存在配体的情况下进行,显示负责基础信号传导活动的自发构象过渡,或者存在激动剂或反向激动剂配体,揭示配体如何改变受体的动力学刺激或抑制信号传导活性。所得到的机械信息对于改进的GPCR靶向药物的设计是有用的。单分子测定法在β2肾上腺素能受体的背景下进行了描述,但可扩展到多种GPCRs。
【背景】GPCR介导本地和远距离的细胞通讯,特别是内分泌系统。例如,细胞对激素如肾上腺素的反应是通过肾上腺素能受体介导的,其中β2肾上腺素能受体(β2AR)是突出的成员。 ...

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