符合规范的GPCR BRET传感器基于非规范性氨基酸的生物对等标记
Maria Kowalski-Jahn1, Hannes Schihada1, Gunnar Schulte2
1Receptor Biology & Signaling, Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Methods in molecular biology (Clifton, N.J.)
|June 5, 2023
概括
研究人员使用遗传密码扩展开发了基于生物发光共振能量转移 (BRET) 的新型传感器. 这些传感器可以实时监测活细胞内的受体动态和相互作用.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 生物物理学的生物物理.
背景情况:
- 膜受体动态对于细胞信号传递至关重要.
- 现有的研究受体构造变化的方法有局限性.
研究的目的:
- 开发一种基于BRET的新型传感器系统,用于分析受体结构动态.
- 为了实时监测活细胞中受体复合体的形成,解离和重新排列.
主要方法:
- 利用遗传密码扩展用于特定地点的非正规氨基酸的结合.
- 工程 BRET 传感器使用 N-终端纳米化酶 (Nluc) 和光标记的非正规氨基酸.
- 应用生物对等标签技术,用于最少侵入性的传感器构造.
主要成果:
- 成功生成了BRET传感器,能够检测分子内 (例如,CRD动态) 和分子间 (例如,二极管动态) 受体重组.
- 证明了在活细胞中随着时间的推移分析受体动态的能力.
- 在微型板格式中验证了传感器系统的实用性.
结论:
- 开发的BRET形态传感器为研究带诱导受体动态提供了一个多功能平台.
- 这种方法有助于研究具有高时间分辨率的各种膜受体.
- 该方法可适应高通量查和药物发现工作.
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