Probing the contacts of a low-affinity substrate with a membrane-embedded transport protein using ¹H-¹³C cross-polarisation magic-angle spinning solid-state NMR

Abstract

Solid-state NMR combined with sample deuteration was used to probe the proximity of the low-affinity substrate D-glucose to its binding site within the Escherichia coli sugar transport protein GalP. Samples of E. coli inner membranes with amplified expression of GalP were incubated in D₂O with D-[¹³C₆]glucose and ¹³C NMR signals from the substrate were assigned in two-dimensional dipolar-assisted rotational resonance (DARR) spectra. The signals were confirmed as representing D-glucose bound to GalP as the peaks were abolished after the substrate was displaced from the specific site with the inhibitor forskolin. The ¹³C chemical shift values for D-[¹³C₆]glucose in solution revealed some differences compared to those for ligand bound to GalP, the differences being most pronounced for positions C1 and C2, and especially for C1 in the α-anomer. ¹³C cross-polarization build-up was measured for C1 and C2 of D-[¹³C₆]glucose and D-[²H₇, ¹³C₆]glucose in GalP membranes suspended in D₂O. The build-up curves for the deuterated substrate reflect intermolecular ¹H-¹³C interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the α-anomer is situated closer to the protein binding site than is the β-anomer, consistent with its relatively high signal intensities and more pronounced chemical shift changes in the 2D-correlation spectra. These results demonstrate the utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins.

Keywords::

• Solid-state NMR
• membrane transport proteins
• ligand-binding
• low affinity
• deuteration

Acknowledgements

This work was funded by the EPSRC [EP/G035695/1] and the EU EDICT consortium (contract 201924). The authors thank Malcolm Levitt (University of Southampton) for support.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.