Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry

Protein Science : a Publication of the Protein Society
N PotierH W Duckworth

Abstract

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form s...Continue Reading

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Citations

Apr 1, 2003·Mass Spectrometry Reviews·Cameron E Forde, Sandra L McCutchen-Maloney
May 28, 2003·Mass Spectrometry Reviews·Lars Konermann, Douglas A Simmons
Jan 18, 2005·Journal of the American Society for Mass Spectrometry·Satoko AkashiYoshifumi Nishimura
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