Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate.
An experimental approach is described in which high resolution 13C 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 degrees C indicated that the alpha- and beta-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 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-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 beta-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.
Patching
Simon G
SG
Astbury Centre for Structural Molecular Biology and Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Henderson
Peter J F
PJ
Herbert
Richard B
RB
Middleton
David A
DA
D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002244:Carbon / Q000737:chemistry; D005576:Colforsin / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D002135:Calcium-Binding Proteins; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005576:Colforsin; D002244:Carbon; D014364:Tryptophan; D005947:Glucose