Selective NMR observation of inhibitor and sugar binding to the galactose-H + symport protein GalP, of Escherichia coli

Abstract

The binding of the transport inhibitor forskolin, synthetically labelled with (13)C, to the galactose-H(+) symport protein GalP, overexpressed in its native inner membranes from Escherichia coli, was studied using cross-polarization magic angle spinning (13)C NMR. (13)C-Labelled D-galactose and D-glucose were displaced from GalP with the singly labelled [7-OCO(13)CH(3)]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 (13)C-labelled forskolin was hampered by interference from natural abundance (13)C in the membranes and so the effectiveness of double-quantum filtration was assessed for the exclusive detection of (13)C spin pairs in sugar (D-[1,2-(13)C(2)]glucose) and inhibitor ([7-O(13)CO(13)CH(3)]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 degrees C) but could be applied in a straightforward way to systems that were kept frozen. At -35 degrees 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 (13)C 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.

Glucose and Glycolysis Are Required for the Successful Infection of Macrophages and Mice by Salmonella enterica Serovar Typhimurium

“…Our data show that inducer exclusion regulates the activity of an S. Typhimurium transporter of a substrate available inside macrophages, which could include lactose, melibiose, and glucose/galactose (33,35,37). In the case of glucose and galactose, the inducer exclusion-dependent transporter is galactose permease (GalP) (2,29,30,33). GalP also transports glucose (39), and its expression is induced by availability of galactose (7).…”

Heteronuclear decoupling interference during symmetry-based homonuclear recoupling in solid-state NMR

“…(17) may be analyzed by splitting the I-spin RF field term into two parts: a large part which conforms to Eq. (17), and a smaller perturbation term which is incorporated into the average Hamiltonian theory. In this section, we avoid this complication by assuming that Eq.…”

Solid‐state NMR for the analysis of high‐affinity ligand/receptor interactions

“…NMR experiments which specifically observe only those coupled sites, such as a double quantum filtered NMR experiment, can be employed to suppress the signals arising from the natural abundance background in the sample. These experiments have been used for selective observation of a 13 C labeled inhibitor forskolin bound to the galactose-H þ symport protein GalP (117). Although signals are visible in the CP-MAS spectra, they arise in a region dominated by strong resonances from the natural abundance 13 C present in lipids and proteins.…”

“…One plausible explanation for this is the poor double quantum excitation and reconversion when performing these experiments on fluid lipid bilayers. Whether this is due to the range of motions present interfering with the double quantum recoupling sequence or the broad CP matching condition leading to coupling to the protons and rapid T 1r relaxation is unclear (117).…”