The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.
A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH 2 -terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures ϳ14°C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH 2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (⌬⌬G Ϸ 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.Although proteins are normally linear polymers of amino acid residues, there are now several examples of ribosomally synthesized polypeptides that are cyclized post-translationally by formation of a normal peptide bond between amino and carboxyl ends via mechanisms that are not well understood (for examples, see Refs. 1-3). These proteins characteristically have unusually high thermal stabilities. Moreover, it has been well established that the deliberate circularization of normally linear proteins by a covalent peptide link between the NH 2 -and COOH-terminal ends provides a route for stabilization of the native structure (4 -6). Thermal stability is an important feature, for example, for use of enzymes as catalysts in industrial applications, and also during long NMR recording times at elevated temperatures. Compared with the engineering of a disulfide bridge between the termini, a peptide link protects against digestion by exo-proteases and is inert in reducing environments, which has implications for therapeutic use of proteins and peptides. The engineering of circular polypeptides is straightforward by the use of inteins.Inteins are protein domains that perform a cis-splicing reaction to excise themselves post-translationally from nascent polypeptide chains (7), forming a new peptide bond between the two remaining parts (the exteins; Fig. 1A). Inteins can be split and expressed as two inactive halves (N-intein, Int N 1 and Cintein, Int C ) that regain activity when brought together (8 -10). This allows production of a protein from two segments that are subsequently ligated together in vitro by the intein in a transsplicing process (11).The expression of a synthetic fusion gene that encodes a protein with the structure Int C -extein-Int N (i.e. containing a circularly permuted split intein; Fig. 1B) can lead to prod...
The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 A [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 A at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 A resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell; side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.
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