Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog. Academic Article uri icon

Overview

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.

publication date

  • June 8, 2020

Research

keywords

  • Amino Acid Transport System X-AG
  • Magnetic Resonance Spectroscopy

Identity

PubMed Central ID

  • PMC7442671

Scopus Document Identifier

  • 85086162920

Digital Object Identifier (DOI)

  • 10.1038/s41589-020-0561-6

PubMed ID

  • 32514183

Additional Document Info

volume

  • 16

issue

  • 9