Fig. 2: ¹⁹F NMR spectra of F₄-TPP⁺ bound to S64V-EmrE in DMPC bilayers.

a Variable-temperature ¹⁹F direct-polarization (DP) spectra measured under 10.5 kHz MAS. The substrate has an isotropic ¹⁹F chemical shift of −106 ppm. The ¹⁹F linewidths and spinning sideband intensities are highly sensitive to temperature. At 245 K, F₄-TPP⁺ is immobilized, as seen by the high sideband intensities, which are simulated (blue) to give the ¹⁹F CSA. In contrast, at 308 K, F₄-TPP⁺ is nearly isotropically mobile. The small sharp peaks at −80 ppm and −116 ppm in the high-temperature spectrum are attributed to residual 4-fluoroiodobenzene and tris(4-fluorophenyl)phosphine from the F₄-TPP⁺ synthesis. b ¹⁹F DP spectrum measured at 285 K under 35 kHz MAS. Spectral deconvolution gives five components, indicating that the ligand experiences a heterogeneous structural environment. ¹³C-¹⁹F cross-polarization (CP) spectrum enhanced three out of the five components, indicating that these species are closest to the ¹³C-labeled protein. c Variable-temperature ¹³C CP MAS spectra of DMPC-bound S64V-EmrE. The spectral intensity decreases with increasing temperature, indicating that the protein becomes more dynamic at higher temperature. d 2D ¹⁹F-¹⁹F correlation spectra of F₄-TPP⁺ with 10 ms mixing, measured under 38 kHz MAS. Exchange peaks are detected at 285 K but not at 265 K, indicating that the exchange is owing to substrate reorientation. e Intensity buildup curves of cross peaks (shown as blue crosses in d) yield an average exchange time constant of 16 ± 2 ms.