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

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-[C-13(6)]glucose and C-13 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 C-13 chemical shift values for D-[C-13(6)]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 alpha-anomer. C-13 cross-polarization build-up was measured for C1 and C2 of D-[C-13(6)]glucose and D-[H-2(7), C-13(6)]glucose in GalP membranes suspended in D2O. The build-up curves for the deuterated substrate reflect intermolecular H-1-C-13 interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the alpha-anomer is situated closer to the protein binding site than is the beta-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.