ARTICLE 1 H, 13 C and 15 N resonance assignments of the chaperone HybE of hydrogenase-2 from Escherichia coli Xuan Shao Jie Lu Bin Xia Changwen Jin Received: 30 December 2008 / Accepted: 23 March 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Escherichia coli HybE is a chaperone of hydrogenase-2 and plays a critical role in coordinating the assembly and export of the HybO and HybC subunits of hydrogenase-2. Previous studies indicated that the quality- control during the assembly and export of the HybO-HybC by the Tat pathway was putatively performed by HybE. However, the molecular basis of the biological function of HybE remains unknown. The structural information is essential in order to obtain functional insights of HybE. Here we report the backbone and sidechain resonance assignments of 1 H, 13 C and 15 N atoms in E. coli HybE. Keywords Escherichia coli Á HybE Á Assignments Á NMR Á Chemical shift Biological context In bacteria, the twin-arginine translocation (Tat) system involves the transmembrane translocation of only fully folded proteins and thus is important for many biological processes (Berks et al. 2005; Sargent 2007). This quality- control machinery is performed by the Tat system chaperones, which bind to the twin-arginine signal peptide to prevent premature targeting of substrates before the biosynthetic processes are completed (DeLisa et al. 2003; Jack et al. 2004). The chaperone HybE from Escherichia coli plays a role during the assembly and translocation of the HybO and HybC subunits of respiratory [NiFe] hydrogenase-2 across the bacterial cytoplasmic membrane by the Tat pathway (Berks et al. 2000; Forzi and Sawers 2007). HybE specif- ically interacts with the twin-arginine signal peptide of the HybO-HybC heterodimer and may assist in the assembly of HybOC (Dubini and Sargent 2003; Jack et al. 2004; But- land et al. 2006). Previous studies showed that only very low hydrogenase-2 activity was observed in a hybE- deletion strain (Jack et al. 2004). To date, studies on Tat chaperones have revealed great structural diversities. Escherichia coli HybE shares \ 12% sequence identity with known Tat chaperones, suggesting a new family of struc- tures of the Tat system. Herein, we report the backbone and sidechain resonance assignments of HybE. Methods and experiments The E. coli hybE gene was cloned into the vector pET-21a (?) (Novagen) and the recombinant plasmid was trans- formed in E. coli BL21 (DE3) strain (Invitrogen) for protein expression. The E. coli cells were cultured in 30 mL of Luria–Bertani (LB) broth medium containing 30 lg/ml of ampicillin at 35°C overnight, and transferred into 1L of LB medium with antibiotics for continuous growth. When the OD 600 reached 0.8, the cell culture was resuspended in 250 ml of M9 minimal medium with ampicillin and 15 NH 4 Cl in the presence or absence of 13 C 6 -glucose for the preparations of 13 C/ 15 N-labeled or X. Shao Á J. Lu Á B. Xia Á C. Jin (&) Beijing Nuclear Magnetic Resonance Center, Peking University, 100871 Beijing, China e-mail: changwen@pku.edu.cn J. Lu Á B. Xia Á C. Jin College of Life Sciences, Peking University, 100871 Beijing, China X. Shao Á B. Xia Á C. Jin College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, China 123 Biomol NMR Assign DOI 10.1007/s12104-009-9157-5