Solid-State NMR Spectroscopy Detects Interactions between Tryptophan Residues of the E. coli Sugar Transporter GalP and the α-Anomer of the D-Glucose Substrate

摘要

An experimental approach is described in which high resolution (~13)C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 ℃ indicated that the α- and β-anomers of D-[1-~(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 ℃, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 ℃ showed a selective interaction between the a-anomer of D-[1-~(13)C]glucose and (~13)C-labels within [(~13)C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the a-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the β-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-~(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.