P. Fsoni, The crystal structure of 57 the C-terminal domain (Stelter et al. 2012), as well as of 58 the 121 N-terminal residues of HpDnaB were determined 59 The latter one was found to consist of 60 a dimer and two further degradation peptide fragments in 61 the asymmetric unit This manuscript 62 presents the solid-state NMR spectroscopic investigation of 63 the N-terminus of HpDnaB as a step towards an integrated 64 structural biology approach aiming at a detailed structural 65 description of the full-length protein. In this context, solid- 66 state NMR studies on a sedimented sample of the full- 67 length protein have been described, Previous biochemical investigations suggest that the 69 N-terminal domain and the linker region play an important 70 role in multimerisation, quaternary state transition and 71 activity of HpDnaB, 2005.

, pylori helicase DnaB (strain 26695) was 78 amplified by PCR (forward 5 0 -caccatggatcatttaaag- 79 catttgcag-3 0 and reverse 5 0 -gcaccatagaaggctttaggaattag-3 0 ) 80 from genomic DNA and inserted into the plasmid pET151/ 81 DTopo (Invitrogen TM ) The resulting vector was intro- 82 duced into E. coli BL21(DE3) cells (One Shot Ò BL21 83 Star TM (DE3) Chemically Competent E. coli, Invitro- 84 gen TM ) and protein overexpression was performed in 85 minimal M9 medium (Studier 2005) containing D-[U-13- 86 C]glucose 2 g L -1 (Cambridge Isotope Laboratories, Inc. 87 CLM-1396-PK) and 15 NH 4 C l 2gL -1 (Sigma-Aldrich Ò 88 299251) as the only nitrogen and carbon sources. After cell 89 lysis by a microfluidization process, 13 C-15 N-DnaB-Nter 90 was purified by Ni 2? -agarose affinity chromatography 91 (Qiagen TM ). The pseudo-affinity tag was subsequently 92 cleaved with the TEV (Tobacco Etch Virus) protease by 93 dialysis. Six additional residues of the tag remain in the 94 sequence (see Fig. 1). 95 For crystallization, 13 C-15 N-DnaB-Nter was concen- 96 trated to 23, 73 Methods and experiments 74 Protein expression and purification, sample 75 preparation 76 The DNA fragment corresponding to the N-terminal 77), and the 98 buffer was exchanged during the concentration step with 99 the final buffer

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