Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.
This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.
Kroon
G J
GJ
Bioson Institute, University of Groningen, The Netherlands.
Grötzinger
J
J
Dijkstra
K
K
Scheek
R M
RM
Robillard
G T
GT
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D004754:Enterobacter / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012486:Salmonella typhimurium / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli