Use of paramagnetic <sup>19</sup>F NMR to monitor domain movement in a glutamate transporter homolog.

Overview

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 ¹⁹F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a ¹⁹F probe via cysteine chemistry and with a Ni²⁺ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of ¹⁹F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-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 ¹⁹F nuclei.