Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

Huang, Y.; Wang, X.; Lv, G.; Razavi, A. M.; Huysman, G. H. M.; Weinstein, H.; Bracken, C.; Eliezer, D.; Boudker, O.

doi:10.1101/832121

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bioRxiv · 10.1101/832121

Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

Huang, Y.·Wang, X.·Lv, G.·Razavi, A. M.·Huysman, G. H. M.·Weinstein, H.·Bracken, C.·Eliezer, D.·Boudker, O.

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Abstract

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 homologue 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 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 19F nuclei.

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Huang, Y., Wang, X., Lv, G., Razavi, A. M., Huysman, G. H. M., Weinstein, H., Bracken, C., Eliezer, D., Boudker, O.. 2019-11-05. Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog. https://doi.org/10.1101/832121

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