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Abstract
Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of 13C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly 13C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.
| Abbreviations | ||
| CP-MAS | = | cross-polarization magic-angle spinning |
| CPPI | = | cross-polarization with polarization inversion |
| MFS | = | major facilitator superfamily |
| OPA | = | Organophoshate:Phosphate Antiporter |
| SDS-PAGE | = | sodium dodecyl sulphate polyacrylamide gel electrophoresis |
| SSNMR | = | solid-state 13C nuclear magnetic resonance |
| SP | = | Sugar Porter |
| TM | = | transmembrane helix |
| WT | = | wild-type |
| Abbreviations | ||
| CP-MAS | = | cross-polarization magic-angle spinning |
| CPPI | = | cross-polarization with polarization inversion |
| MFS | = | major facilitator superfamily |
| OPA | = | Organophoshate:Phosphate Antiporter |
| SDS-PAGE | = | sodium dodecyl sulphate polyacrylamide gel electrophoresis |
| SSNMR | = | solid-state 13C nuclear magnetic resonance |
| SP | = | Sugar Porter |
| TM | = | transmembrane helix |
| WT | = | wild-type |