Selective NMR observation of inhibitor and sugar binding to the galactose-H⁺ symport protein GalP of <i>Escherichia coli</i>

The binding of the transport inhibitor forskolin, synthetically labelled with 13C, to the galactose-H+ symport protein GalP, overexpressed in its native inner membranes from Escherichia coli, was studied using cross-polarization magic angle spinning 13C NMR. 13C-Labelled d-galactose and d-glucose were displaced from GalP with the singly labelled [7-OCO13CH3]forskolin and were not bound to any alternative site within the protein, demonstrating that any multiple sugar binding sites are not simultaneously accessible to these sugars and the inhibitor within GalP. The observation of singly 13C-labelled forskolin was hampered by interference from natural abundance 13C in the membranes and so the effectiveness of double-quantum filtration was assessed for the exclusive detection of 13C spin pairs in sugar (d-[1,2-13C2]glucose) and inhibitor ([7-O13CO13CH3]forskolin) bound to the GalP protein. The solid state NMR methodology was not effective in creating double-quantum selection of ligand bound with membranes in the ‘fluid’ state (approx. 2°C) but could be applied in a straightforward way to systems that were kept frozen. At −35°C, double-quantum filtration detected unbound sugar that was incorporated into ice structure within the sample, and was not distinguished from protein-bound sugar. However, the method detected doubly labelled forskolin that is selectively bound only to the transport system under these conditions and provided very effective suppression of interference from natural abundance 13C background. These results indicate that solid state NMR methods can be used to resolve selectively the interactions of more hydrophobic ligands in the binding sites of target proteins.