Abstract
The heat shock protein ClpB in Escherichia coli is a protein-activated ATPase and consists of two proteins with sizes of 93 and 79 kDa. By polymerase chain reaction-aided site-directed mutagenesis, both the proteins have been shown to be encoded by the same reading frame of the clpB gene, the 93-kDa protein (ClpB93) from the 5'-end AUG translational initiation site and the 79-kDa protein (ClpB79) from the 149th codon (an internal GUG start site). Both the purified ClpB93 and ClpB79 proteins behave as tetrameric complexes with a very similar size of about 350 kDa upon gel filtration on a Superose-6 column. Both appear to be exclusively localized to the cytosol of E. coli. Both show inherent ATPase activities and have an identical Km of 1.1 mM for ATP. The ATPase activity of ClpB93 is as markedly stimulated by proteins, including casein and insulin, as that of wild-type ClpB, but the same proteins show little or no effect on ClpB79. Because ClpB79 lacks the 148 N-terminal sequence of ClpB93 but retains the two consensus sequences for adenine nucleotide binding, the N-terminal portion appears to contain a site(s) or domain(s) responsible for protein binding. Furthermore, ClpB79 is capable of inhibiting the casein-activated ATPase activity of ClpB93 in a dose-dependent manner but without any effect on its inherent ATPase activity. In addition, ClpB93 mixed with differing amounts of ClpB79 behave as tetrameric molecules, although its protein-activated ATPase activity is gradually reduced. These results suggest that tetramer formation between ClpB93 and ClpB79 may be responsible for the inhibition of the activity.
Highlights
The heatshock protein ClpB in Escherichia coli is a stimulate ATP-hydrolyzing activity of the enzyme [7]
The ATPase activity homology, each of which contains a consensus sequence for of ClpB93 is as markedly stimulated by proteins, in- an adeninenucleotide binding site.ClpB, which alsois a heat cluding casein and insulin,as that of wild-type ClpB, shock protein [17, 18], has recentlybeen purified and shown butthesameproteinsshowlittleor no effecton to be a protein-activated ATPase [19]
Fragments IV and V were gap-filled by Klenow and ligated into ing the mutated plasmids, ClpB93 and ClpB79 were purified by the pBS, and the recombinant plasmidwsere maintained in E. coli strain same protocol used for the purification of wild-type ClpB [19]
Summary
Base substitution amounts of the cells were harvested, boiled, and electrophoresed in. Assays-ATPase activity was assayed by incubating the reaction mixtures (0.1 ml) containing appropriate amounts of the mutated ClpB proteins, 5 mM ATP, 100 mM Tris-HCI (pH 8), 10 mMMgC12, 1mM dithiothreitol, and 1 mM EDTA [19]. Fragment IV was synthesized using primers mutant (MC1000AclpB::Km’); b, the cell harboringpBS-ClpB; c, pBS-. Fragments IV and V were gap-filled by Klenow and ligated into ing the mutated plasmids, ClpB93 and ClpB79 were purified by the pBS, and the recombinant plasmidwsere maintained in E. coli strain same protocol used for the purification of wild-type ClpB [19]. Structure and ATPase Activities of ClpB79 and ClpB93To determine thestructural organization of ClpB79 and ClpB93, each of the proteins was purified from the crude extracts of MC1079 and MC1093, respectively,using the same differing amounts of ClpB79 alone were subtracted from those with purification protocol used for the wild-type ClpB [19]. We chromatographed the purified proteins on a Superose-6 col-
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