Enzyme IIB bcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: Backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy
✍ Scribed by Eiso Ab; Gea K. Schuurman-Wolters; Milton H. Saier; Jonathan Reizer; Michel Jacuinod; Peter Roepstorff; Klaas Dijkstra; Ruud M. Scheek; George T. Robillard
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 2008
- Tongue
- English
- Weight
- 828 KB
- Volume
- 3
- Category
- Article
- ISSN
- 0961-8368
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✦ Synopsis
Abstract
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^Cα 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^Cα and ^13^CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3‐dimensional ^1^Ha‐^13^Cα‐^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^Hα and ^13^Cα 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 β‐sheet and 5 α‐helices. In the wild‐type enzyme IIB^cellobiose^, the catalytic residue appears to be located at the end of a β‐strand.