1h, 13c, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli

1H, 13C, and 15N NMR assignments of the backbone atoms and -carbons have been madefor liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with226 amino acid residues and a molecular weight of 24,935 Da. GlnBP is a periplasmicbinding protein which plays an essential role in the active transport of L-glutamine throughthe cytoplasmic membrane. The assignments have been obtained from three-dimensionaltriple-resonance NMR experiments on a 13C,15N uniformly labeled sample as well asspecifically labeled samples. Results from the 3D triple-resonance experiments, HNCO,HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are themain sources used to make the resonance assignments. Other 3D experiments, such asHNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirmthe resonance assignments and to extend connections where resonance peaks are missing insome of the experiments mentioned above. We have assigned more than 95% of thepolypeptide backbone resonances of GlnBP. The result of the standard manual assignment isin agreement with that predicted by an automated probabilistic method developed in ourlaboratory. A solution secondary structure of the GlnBP–Gln complex has beenproposed based on chemical shift deviations from random coil values. Eight -helices and10 -strands are derived using the Chemical Shift Index method.