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 struct ure determination of the A domain of the Escherichia coli mannitol tra nsport protein, enzyme-II(mtl). The backbone resonances were partially assigned using three-dimensional heteronuclear H-1 NOE H-1-N-15 singl e-quantum coherence (N-15 NOESY-HSQC) spectroscopy and three-dimension al heteronuclear H-1 total correlation H-1-N-15 single-quantum coheren ce (N-15 TOCSY-HSQC) spectroscopy on uniformly ''N enriched protein. T riple-resonance experiments on uniformly N-15/C-13 enriched protein we re necessary to complete the backbone assignments, due to overlapping H-1 and N-15 frequencies. Data obtained from three-dimensional H-1-N-1 5-C-13alpha correlation experiments (HNCA and HN(CO)CA), a three-dimen sional H-1-N-15-(CO)-C-13 correlation experiment (HNCO), and a three-d imensional H-1alpha-C-13alpha-(CO)-C-13 correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of vi rtually all observed backbone resonances. Determination of the seconda ry structure of IIA(mtl) is based upon NOE information from the N-15 N OESY-HSQC and the H-1alpha and C-13alpha secondary chemical shifts. Th e resulting secondary structure is considerably different from that re ported for IIA(glc) of E. coli and Bacillus subtilis determined by NMR and X-ray.