ThednaCprotein ofEscherichia coli. Purification, physical properties and interaction withdnaBprotein

Abstract

E. coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S. Wickner, I. Berkower, M. Wright, and J. Hurwitz (1973) Proc. Natl. Acad. Sci. USA 70, 2369-2373]. Purification was achieved by taking advantage of the hydrophobic interaction ofdnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent. A sedimentation coefficient for the dnaC protein of 2.6S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient cen-trifugation. A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel. In the presence of ATP the dnaC protein forms a complex with dnaB protein [S. Wickner and J. Hurwitz (1975) Proc. Natl. Acad. Sci. USA 72, 921-925]. For the dnaB–dnaC complex a sedimentation coefficient of 14.5S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000. The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel. It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000.