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Precise limits of the N-terminal domain of DnaB helicase determined by NMR spectroscopy

Journal Article


Abstract


  • Two separate N-terminal fragments of the 470-amino-acid Escherichia coli DnaB helicase, comprising residues 1-142 and 1-161, were expressed in E. coli. The proteins were extracted in a soluble fraction, purified, and characterised physically. In contrast to the full-length protein, which is hexameric, both fragments exist as monomers in solution, as demonstrated by sedimentation equilibrium measurements. CD spectroscopy was used to confirm that the 161-residue fragment is highly structured (mostly α-helical) and undergoes reversible thermal denaturation. The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by assignment of resonances from flexible residues in NMR spectra. The 1H NMR signals of the flexible residues are located at random coil chemical shifts, and their linewidths are significantly narrower than those of the structured core, indicating complete disorder and increased mobility on the nanosecond time scale. The results support the idea of a flexible hinge region between the N- and C-terminal domains of the native hexameric DnaB protein.

Publication Date


  • 1997

Citation


  • Miles, C. S., Weigelt, J., Stamford, N. P. J., Dammerova, N., Otting, G., & Dixon, N. E. (1997). Precise limits of the N-terminal domain of DnaB helicase determined by NMR spectroscopy. Biochemical and Biophysical Research Communications, 231(1), 126-130. doi:10.1006/bbrc.1997.6059

Scopus Eid


  • 2-s2.0-0031550557

Start Page


  • 126

End Page


  • 130

Volume


  • 231

Issue


  • 1

Abstract


  • Two separate N-terminal fragments of the 470-amino-acid Escherichia coli DnaB helicase, comprising residues 1-142 and 1-161, were expressed in E. coli. The proteins were extracted in a soluble fraction, purified, and characterised physically. In contrast to the full-length protein, which is hexameric, both fragments exist as monomers in solution, as demonstrated by sedimentation equilibrium measurements. CD spectroscopy was used to confirm that the 161-residue fragment is highly structured (mostly α-helical) and undergoes reversible thermal denaturation. The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by assignment of resonances from flexible residues in NMR spectra. The 1H NMR signals of the flexible residues are located at random coil chemical shifts, and their linewidths are significantly narrower than those of the structured core, indicating complete disorder and increased mobility on the nanosecond time scale. The results support the idea of a flexible hinge region between the N- and C-terminal domains of the native hexameric DnaB protein.

Publication Date


  • 1997

Citation


  • Miles, C. S., Weigelt, J., Stamford, N. P. J., Dammerova, N., Otting, G., & Dixon, N. E. (1997). Precise limits of the N-terminal domain of DnaB helicase determined by NMR spectroscopy. Biochemical and Biophysical Research Communications, 231(1), 126-130. doi:10.1006/bbrc.1997.6059

Scopus Eid


  • 2-s2.0-0031550557

Start Page


  • 126

End Page


  • 130

Volume


  • 231

Issue


  • 1