Open AccessUse of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog
Author(s) -
Yun Huang,
Xiaoyu Wang,
Guohua Lv,
Asghar M. Razavi,
Gerard H. M. Huysmans,
Harel Weinstein,
Clay Bracken,
David Eliezer,
Olga Boudker
Publication year - 2020
Publication title -
nature chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.412
H-Index - 216
eISSN - 1552-4469
pISSN - 1552-4450
DOI - 10.1038/s41589-020-0561-6
Subject(s) - chemistry , transporter , fluorine 19 nmr , biophysics , nuclear magnetic resonance spectroscopy , histidine , intracellular , extracellular , structural biology , cysteine , nuclear magnetic resonance , amino acid , biochemistry , stereochemistry , biology , physics , gene , enzyme
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 homolog 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 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 19 F nuclei.