Expanding the membrane-protein NMR toolkit

Abstract

An NMR method to monitor conformational states of challenging large protein targets is described. The method, which can be used to evaluate distances between two labels and to measure conformational exchange rates, revealed an unanticipated outward-facing state in a glutamate transporter.

Fig. 1 ∣. ¹⁹F PRE NMR as a complement to FRET for evaluating conformational states.

a, Top: view of the GltPh homotrimer from the extracellular side. The scaffold domains are shown in grey and the transport domains in purple. Substrate aspartate and Na⁺ are shown in spacefill. Bottom: Side view of two GltPh subunits, indicating one of the labeling positions used in FRET studies. The ruler below indicates the distances between probes suitable for FRET measurements. b, Structures of the three GltPh conformational states observed in this NMR study (PDB IDs 3V8G, 6UWL, and 6UWF for the IFS, iOFS, and OFS, respectively). In the right are expanded views of the labeling positions. (Here, wild-type residues are shown; in the NMR study, residues were mutated to histidine or cysteine to generate the Ni²⁺-binding or ¹⁹F-labeling site, respectively.) The ruler below indicates the distances between probe sites that can be measured PRE NMR.