BACKBONE ASSIGNMENTS AND SECONDARY STRUCTURE OF THE ESCHERICHIA-COLI ENZYME-II MANNITOL A-DOMAIN DETERMINED BY HETERONUCLEAR 3-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-II(mtl). The backbone resonances were partially assigned using three-dimensional heteronuclear H-1 NOE H-1-N-15 single-quantum coherence (N-15 NOESY-HSQC) spectroscopy and three-dimensional heteronuclear H-1 total correlation H-1-N-15 single-quantum coherence (N-15 TOCSY-HSQC) spectroscopy on uniformly ''N enriched protein. Triple-resonance experiments on uniformly N-15/C-13 enriched protein were necessary to complete the backbone assignments, due to overlapping H-1 and N-15 frequencies. Data obtained from three-dimensional H-1-N-15-C-13alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional H-1-N-15-(CO)-C-13 correlation experiment (HNCO), and a three-dimensional H-1alpha-C-13alpha-(CO)-C-13 correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIA(mtl) is based upon NOE information from the N-15 NOESY-HSQC and the H-1alpha and C-13alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIA(glc) of E. coli and Bacillus subtilis determined by NMR and X-ray.