POLYPEPTIDE BACKBONE RESONANCE ASSIGNMENTS AND SECONDARY STRUCTURE OF BACILLUS-SUBTILIS ENZYME-IIIGLC DETERMINED BY 2-DIMENSIONAL AND 3-DIMENSIONAL HETERONUCLEAR NMR-SPECTROSCOPY

The enzyme III(glc)-like domain of Bacillus subtilis II(glc.), (III(glc.), 162 residues, 17.4 kDa) has been cloned and overexpressed in Escherichia coli. Sequence-specific assignment of the backbone H-1 and N-15 resonances has been carried out with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional (3D) NMR spectroscopy. Amide proton solvent exchange rate constants have been determined from a series of H-1-N-15 heteronuclear single-quantum coherence (HSQC) spectra acquired following dissolution of the protein in D2O. Major structural features of III(glc) have been inferred from the pattern of short-, medium- and long-range NOEs in 3D heteronuclear H-1 nuclear Overhauser effect H-1-N-15 multiple-quantum coherence (3D NOESY-HMQC) spectra, together with the exchange rate constants. III(glc) contains three antiparallel beta-sheets comprised of eight, three, and two beta-strands. In addition, five beta-bulges were identified. No evidence of regular helical structure was found. The N-terminal 15 residues of the protein appear disordered, which is consistent with their being part of the Q-linker that connects the C-terminal enzyme III(glc)-like domain to the membrane-bound II(glc) domain. Significantly, two histidine residues, His 68 and His 83, which are important for phosphotransferase function, are found from NOE measurements to be in close proximity at the ends of adjacent strands in the major beta-sheet.