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. (March 2013)

• Record Type: Journal Article
• Authors: PatchinG, Simon G.; Henderson, Peter J. F.; Sharples, David J.; Middleton, David A.
• Is Part Of: Molecular membrane biology. Volume 30:Number 2(2013)
• Page Start: 129
• Page End: 137
• Publication Date: 2013-03
• Subjects: Membranes (Biology) -- Periodicals; Biochemistry -- Periodicals; Cell membranes -- Periodicals; Molecular biology -- Periodicals; 571.64
• DOI: 10.3109/09687688.2012.743193
• Languages: English
• ISSNs: 0968-7688

Abstract

<abstract> <title>Abstract</title> <p>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 <italic>Escherichia coli</italic> sugar transport protein GalP. Samples of <italic>E. coli</italic> inner membranes with amplified expression of GalP were incubated in D<sub>2</sub>O with D-[<sup>13</sup>C<sub>6</sub>]glucose and <sup>13</sup>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 <sup>13</sup>C chemical shift values for D-[<sup>13</sup>C<sub>6</sub>]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. <sup>13</sup>C cross-polarization build-up was measured for C1 and C2 of D-[<sup>13</sup>C<sub>6</sub>]glucose and D-[<sup>2</sup>H<sub>7</sub>, <sup>13</sup>C<sub>6</sub>]glucose in GalP membranes suspended in D<sub>2</sub>O. The build-up curves for the deuterated substrate reflect intermolecular <sup>1</sup>H-<sup>13</sup>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.</p> </abstract>