Single-molecule FRET (smFRET) can visualize conformational dynamics of individual ion channels in lipid bilayers of defined composition. Dynamic and distance measurements from smFRET, combined with single channel recordings, can provide previously unattainable direct mechanistic insights into ion channel function and modulation. smFRET measurements require site-specific fluorophore labeling between two distinct sites, which is a major challenge for multimeric ion channels. This chapter aims to provide a step-by-step protocol: (1) to design concatemeric constructs with only two cysteine residues within a homotetrameric channel; (2) to express, purify, label, and reconstitute channel proteins; (3) to perform smFRET imaging on channel proteins in liposomes with an objective-based Total Internal Reflection (TIRF) microscope; and finally (4) to analyze the FRET distributions and dynamics that reflect the dynamic conformational transitions of ion channels in membranes.
Membrane protein Ion channel Conformational dynamics Fluorophore labeling Single-molecule FRET TIRF
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The authors would like to thank Dr. Jonathan Silva for his help at early state in constructing the TIRF microscope for smFRET measurements, and Dr. William Stump for his help in developing the script converting imaging files. The chapter was written by S.W., and edited by J.B. and C.N.
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