Experimental and theoretical distance dependence of tmFRET with the membrane. (A) The cryoEM structure of the ARD of TRPV1 is shown oriented relative to the expected intracellular surface of the plasma membrane. The three amino acids replaced by L-ANAP in this study are shown as cyan spheres, with the distance of each from the surface of the plasma membrane indicated on the abscissa. (B) Relationship between the FRET efficiency and distance from the surface of the plasma membrane. The points represent the experimentally determined FRET efficiency calculated from quenching of L-ANAP incorporated into each position by Co²⁺ bound to C18-NTA on the plasma membrane. The position of the membrane surface and its uncertainty were estimated as the mean and standard deviation of the z coordinate of the charged atoms of amino acids (K431, R432, R491, R557, and K579) in the cryoEM structure (Cao et al., 2013; Liao et al., 2013). The error bars for FRET efficiency are the SEM of the quenching measurements. The smooth curve is the theoretical efficiency calculated based on a model of FRET in two dimensions (Fung and Stryer, 1978).