PRECISE LIMITS OF THE N-TERMINAL DOMAIN OF DNAB HELICASE DETERMINED BY NMR-SPECTROSCOPY

Two separate N-terminal fragments of the 470-amino-acid Escherichia co li 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 spectrosco py was used to confirm that the 161-residue fragment is highly structu red (mostly alpha-helical) and undergoes reversible thermal denaturati on. The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by ass ignment of resonances from flexible residues in NMR spectra. The H-1 N MR signals of the flexible residues are located at random coil chemica l shifts, and their linewidths are significantly narrower than these o f the structured core, indicating complete disorder and increased mobi lity on the nanosecond time scale, The results support the idea of a f lexible hinge region between the N- and C-terminal domains of the nati ve hexameric DnaB protein. (C) 1997 Academic Press.