Protein Science, Vol 3, Issue 2 282-290, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Enzyme IIB(cellobiose) of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: Backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy

E. AB, G. K. SCHUURMAN-WOLTERS, M. H. SAIER, J. REIZER, M. JACUINOD, P. ROEPSTORFF, K. DIJKSTRA, R. M. SCHEEK and G. T. ROBILLARD
The Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIB(cellobiose) of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone (1)H, (15)N, and (13)C{alpha} resonances, and the HN(CA)CO and HNCO experiments, correlating backbone (1)H,(15)N and (13)CO resonances. Heteronuclear (1)H-NOE (1)H-(15)N single quantum coherence ((15)N-NOESY-HSQC) spectroscopy and heteronuclear (1)H total correlation (1)H-(15)N single quantum coherence ((15)N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping (13)C{alpha} and (13)CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional (1)H{alpha}-(13)C{alpha}-(13)CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed (1)H{alpha} and (13)C{alpha} chemical shifts from their random coil values and (1)H-NOE information from the (15)N-NOESY-HSQC. These data show that enzyme IIB(cellobiose) consists of a 4-stranded parallel {beta}-sheet and 5 {alpha}-helices. In the wild-type enzyme IIB(cellobiose), the catalytic residue appears to be located at the end of a {beta}-strand.
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