We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with N-15 and C-13 to overcome spectral overlap. Complete assignments were obtained for the backbone H-1, N-15 and C-13 resonances, using three-dimensional heteronuclear H-1 NOE (H-N)-H-1-N-15 multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation (H-N)-H-1-N-15 multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on N-15-enriched HPr and an additional three-dimensional triple-resonance (HN-N-C)-H-1-N-15-C-13-alpha correlation spectroscopy (HNCA) experiment on C-13, N-15-enriched HPr. Many of the sequential backbone H-1 assignments, as derived from two-dimensional NMR studies [Klevit, R. E., Drobny, G. P. & Waygood, E. B. (1986) Biochemistry 25,7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.
|Tijdschrift||European Journal of Biochemistry|
|Nummer van het tijdschrift||3|
|Status||Published - 1-feb-1992|