Use of Paramagnetic (19)F NMR to Monitor Domain Movement in a Glutamate Transporter

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel (19)F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a (19)F probe via cysteine chemistry and with a Ni(2+) ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of (19)F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of (19)F nuclei.