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

Nature Chemical Biology
Yun HuangOlga Boudker

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.

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Citations

Jul 16, 2020·Nature Chemical Biology·Ricky C Cheng, Merritt Maduke
Oct 1, 2020·Journal of Biomolecular NMR·Jan H OverbeckRemco Sprangers
Oct 8, 2020·Nature Communications·Tina R MatinSimon Scheuring
Jan 28, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Li Shi, Naixia Zhang
Apr 30, 2021·Chemical Communications : Chem Comm·Jia-Liang ChenXun-Cheng Su
May 5, 2021·Chembiochem : a European Journal of Chemical Biology·Kim BartelsChristian Löw
Jun 16, 2021·Current Opinion in Structural Biology·Louis-Philippe Picard, Robert Scott Prosser

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Methods Mentioned

BETA
NMR
atomic force microscopy
chemical exchange
FRET
RSMR
size exclusion chromatography
fluorescence imaging

Software Mentioned

Appion
MDTraj
NAMD
Modeller
MotionCorr2
RELION
Phenix
UCSF Chimera
SerialEM
Molprobity

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