title: 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 D(2)O with D-[(13)C(6)]glucose and (13)C 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 (13)C chemical shift values for D-[(13)C(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 α-anomer. (13)C cross-polarization build-up was measured for C1 and C2 of D-[(13)C(6)]glucose and D-[(2)H(7), (13)C(6)]glucose in GalP membranes suspended in D(2)O. The build-up curves for the deuterated substrate reflect intermolecular (1)H-(13)C 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.
authors.affiliation: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, UK. s.g.patching@leeds.ac.uk
authors.forename: Simon G
authors.initials: SG
authors.lastname: Patching
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Henderson
authors.forename: David J
authors.initials: DJ
authors.lastname: Sharples
authors.forename: David A
authors.initials: DA
authors.lastname: Middleton
mesh_terms: D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D005576:Colforsin / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
chemical_list: D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005576:Colforsin; D005947:Glucose