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.
Subject(s)
Bacterial Proteins/chemistry , DNA Helicases/chemistry , Protein Conformation , Circular Dichroism , DnaB Helicases , Escherichia coli/enzymology , Magnetic Resonance Spectroscopy , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The DnaB protein is the major replicative DNA helicase in Escherichia coli. It hydrolyzes ATP to promote its translocation in the 5' to 3' direction on single-stranded DNA templates, facilitating the separation of strands of duplex DNA in its path. This places it on the lagging strands at replication forks during chromosomal DNA replication. Electron microscopic images of negatively stained DnaB protein have been studied and processed to produce a three-dimensional reconstruction of the protein oligomer at 2.7 nm resolution. While it is known that the native protein is a complex of six identical 52-kDa subunits, the specimen shows threefold rather than sixfold symmetry, with three outer stain-excluding regions surrounding another six, more massive, lobules. There is a channel through the particle that appears fully open on both sides. Based on these results, a structural model for the oligomer is presented, and functional implications are considered.
