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

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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.