Fig. 4: Protein–substrate distance measurement using ¹H-¹⁹F and ¹³C-¹⁹F REDOR experiments.

a Representative S₀ (red) and ΔS (blue) 2D REDOR-hNH correlation spectra, measured with mixing times of 1.68 ms and 3.78 ms. Assignment is shown for selected peaks in the S₀ spectrum. More difference peaks are observed in the 3.78 ms ΔS spectrum than the 1.68 ms ΔS spectrum due to the detection of H^N sites further away from the substrate at longer mixing times. b Representative ¹H-¹⁹F REDOR S/S₀ dephasing curves with best-fit simulations. Fast and slow dephasing, corresponding to short and long distances, are shown in blue and black, respectively. c Cα secondary chemical shifts (gray bars) of F₄-TPP⁺ bound EmrE, indicating the four TM α-helices separated by short loops. Residues whose H^N atoms show difference signals in the 2D REDOR-hNH spectra are indicated by magenta circles at the bottom. Best-fit H^N-F distances are indicated by blue circles. Residues in TM3A, TM3B, and TM1A display short distances to F₄-TPP⁺. d Topology of the eight TM helices in the dimeric EmrE, with monomer A helices shown in orange and monomer B helices shown in blue. e 1D ¹³C-¹⁹F REDOR S₀ and ΔS spectra, coadded from spectra recorded with mixing times of 0.92, 2.0, 3.0, and 4.5 ms. The ΔS spectrum is scaled up by 32-fold with respect to the S₀ spectrum to better display the signals of substrate-proximal ¹³C sites. Note the preferential increase of aromatic ¹³C intensities in the 100–160 ppm region in the ΔS spectrum compared to the control S₀ spectrum. This is consistent with the dominance of aromatic residues at the binding site. Selected peaks are assigned based on the chemical shifts assigned from the 3D correlation spectra (Supplementary Table 1).