title: Precise limits of the N-terminal domain of DnaB helicase determined by NMR spectroscopy.
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 alpha-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.
authors.affiliation: Centre for Molecular Structure and Function, Research School of Chemistry, Australian National University, Canberra, Australia.
authors.forename: C S
authors.initials: CS
authors.lastname: Miles
authors.forename: J
authors.initials: J
authors.lastname: Weigelt
authors.forename: N P
authors.initials: NP
authors.lastname: Stamford
authors.forename: N
authors.initials: N
authors.lastname: Dammerova
authors.forename: G
authors.initials: G
authors.lastname: Otting
authors.forename: N E
authors.initials: NE
authors.lastname: Dixon
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004265:DNA Helicases / Q000737:chemistry*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D004265:DNA Helicases; D053488:DnaB Helicases