Mass spectrometric study of the Escherichia coli repressor proteins, Iclr and GclR, and their complexes with DNA

In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR-lik e proteins, now known as the IclR family, which can be identified by a cons erved C-terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although puri fication is straightforward, both the IclR and GclR proteins are difficult to manipulate, requiring high salt (up to 0.6 M KCI) for solubility. With t he advent of nanospray ionization, we could transfer the proteins into much higher concentrations of volatile buffer than had been practical with ordi nary electrospray. In 0.5 M ammonium bicarbonate buffer, both proteins were stable as tetramers, with a small amount of dimer. In a separate experimen t, 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, al though IclR does not regulate the gel gene. We designed a 29 bp synthetic D NA to which IclR and GclR bind, and with which we were able to form noncova lent DNA-protein complexes for further mass spectrometry analysis. These co mplexes were far more stable than the proteins alone, and we have evidence of a stoichiometry which has not been described previously with (protein mo nomer : dsDNA) = (4 : 1).