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Mass spectrometric study of the Escherichia coli repressor proteins, IcIR and GcIR, and their complexes with DNA

¹ Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
² Department of Physics, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada

Reprint requests to: Harry W. Duckworth, Department of Chemistry, 531 Parker Building, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada; e-mail: hdckwth{at}cc.umanitoba.ca (204) 474-7608.

In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR-like proteins, now known as the IclR family, which can be identified by a conserved C-terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although purification is straightforward, both the IclR and GclR proteins are difficult to manipulate, requiring high salt (up to 0.6 M KCl) for solubility. With the advent of nanospray ionization, we could transfer the proteins into much higher concentrations of volatile buffer than had been practical with ordinary electrospray. In 0.5 M ammonium bicarbonate buffer, both proteins were stable as tetramers, with a small amount of dimer. In a separate experiment, we found that IclR protein selected from a random pool a sequence which matched exactly that of the presumed binding region of the GclR protein, although IclR does not regulate the gcl gene. We designed a 29 bp synthetic DNA to which IclR and GclR bind, and with which we were able to form noncovalent DNA-protein complexes for further mass spectrometry analysis. These complexes were far more stable than the proteins alone, and we have evidence of a stoichiometry which has not been described previously with (protein monomer : dsDNA) = (4 : 1).

Keywords: Protein-DNA interaction; noncovalent; nanospray ionization; IcIR family protein; time-of-flight mass spectrometry

Abbreviations: PCR, polymerase chain reaction • EMSA, electrophoretic mobility shift assay • TPP, thiamine pyrophosphate (cocarboxylase) • DTT, dithiothreitol • ESI, electrospray ionization • TOF MS, time-of-flight mass spectrometry • SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis • MS/MS, tandem mass spectrometry (for sequencing) • FWHM, full width half maximum (error measurement).

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