Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog.
| Publication Type | Academic Article |
| Authors | Huang Y, Wang X, Lv G, Razavi A, Huysmans G, Weinstein H, Bracken C, Eliezer D, Boudker O |
| Journal | Nat Chem Biol |
| Volume | 16 |
| Issue | 9 |
| Pagination | 1006-1012 |
| Date Published | 06/08/2020 |
| ISSN | 1552-4469 |
| Keywords | Amino Acid Transport System X-AG, Magnetic Resonance Spectroscopy |
| 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 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. |
| DOI | 10.1038/s41589-020-0561-6 |
| PubMed ID | 32514183 |
| PubMed Central ID | PMC7442671 |