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 beta-carbons have been made for liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with 226 amino acid residues and a molecular weight of 24935 Da. GlnBP is a periplasmic binding protein which plays an essential role in the active transport of L-glutamine through the cytoplasmic membrane. The assignments have been obtained from three-dimensional triple-resonance NMR experiments on a 13C, 15N uniformly labeled sample as well as specifically 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 the main sources used to make the resonance assignments. Other 3D experiments, such as HNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirm the resonance assignments and to extend connections where resonance peaks are missing in some of the experiments mentioned above. We have assigned more than 95% of the polypeptide backbone resonances of GlnBP. The result of the standard manual assignment is in agreement with that predicted by an automated probailistic method developed in our laboratory. A solution secondary structure of the GlnBP-Gln complex has been proposed based on chemical shift deviations from random coil values. Eight alpha-helices and 10 beta-strands are derived using the Chemical Shift Index method.
Yu
J
J
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213-2683, USA.
Simplaceanu
V
V
Tjandra
N L
NL
Cottam
P F
PF
Lukin
J A
JA
Ho
C
C
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary
D002247:Carbon Isotopes; D002352:Carrier Proteins; D009587:Nitrogen Isotopes; C037853:glutamine transport proteins; D005973:Glutamine; D006859:Hydrogen