Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog

• Published in: Nature chemical biology Vol. 16; no. 9; pp. 1006 - 1012
• Main Authors: Huang, Yun, Wang, Xiaoyu, Lv, Guohua, Razavi, Asghar M., Huysmans, Gerard H. M., Weinstein, Harel, Bracken, Clay, Eliezer, David, Boudker, Olga
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.09.2020; Nature Publishing Group
• Subjects: 631/535/878; 631/57; 631/92/577; Accessibility; Binding sites; Biochemical Engineering; Biochemistry; Bioorganic Chemistry; Cell Biology; Chelation; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Glutamic acid transporter; Histidine; Homology; Magnetic resonance spectroscopy; Membrane proteins; NMR; NMR spectroscopy; Nuclear magnetic resonance; Nuclei; Protein transport; Proteins; Substrates
• ISSN: 1552-4450; 1552-4469
• DOI: 10.1038/s41589-020-0561-6

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. A 19 F-NMR-based method monitoring the conformational dynamics of the glutamate transporter GltPh identified one inward- and two outward-facing states, including one unanticipated outward-facing state that was characterized by cryo-EM.