Black-lipid-membrane platform with diffusion suppression for confocal single-molecule FLIM-FRET analysis of gated membrane receptors and transporters (A01)

具有扩散抑制功能的黑脂质膜平台，用于门控膜受体和转运蛋白的共焦单分子 FLIM-FRET 分析 (A01)

• 批准号: 279179418
• 金额: --
• 依托单位: Arbeitsgruppe Mikroskopie-Methodik
• 依托单位国家: 德国
• 项目类别: CRC/Transregios
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/279179418?language=en
• 关键词: Black; lipid; membrane; platform; diffusion

G-protein coupled membrane receptors neurotensin-1 (NTSR-1) are expressed in bacteria, purified and labeled with two fluorophores for single-molecule Förster resonance energy transfer (smFRET). Reconstituted in specific liposomes, NTSR-1 or HCN2 ion channels will be fused with a planar black-lipid-membrane (BLM). Dynamic conformational changes are studied using time-resolved confocal as well as widefield smFRET microscopy in microfluidic chips with adjustable membrane potentials. Diffusion of membrane proteins in the BLM will be suppressed by an Annexin-V network for extended observation times.

G蛋白偶联膜受体神经降压素-1（NTSR-1）在细菌中表达，纯化并用两个荧光团标记，用于单分子Förster共振能量转移（smFRET）。NTSR-1或HCN 2离子通道在特定脂质体中重构后，将与平面黑脂质膜（BLM）融合。动态构象变化的研究，使用时间分辨共聚焦以及宽场smFRET显微镜在微流控芯片与可调膜电位。膜蛋白在BLM中的扩散将被膜联蛋白-V网络抑制以延长观察时间。