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1 Department of Chemistry, University of Wollongong, New South Wales 2522, Australia
2 Research School of Chemistry, Australian National University, Australian Capital Territory 0200, Australia
Reprint requests to: Dr. Jennifer Beck, Department of Chemistry, University of Wollongong, NSW 2522, Australia; e-mail: jbeck{at}uow.edu.au; fax: 61-2-42-214287.
Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus–Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus–Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus–Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 x 10-9 M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of 
2 x 10-9 M for Tus where A173 was unchanged. This is the first example in which ESI-MS has been used to compare binding affinities of a DNA-binding protein with mutant proteins for specific DNA recognition sequences. It was also possible to estimate the strength of the interaction between Tus and a DNA sequence (TerH) that had been identified by database searching.
Keywords: Tus; DNA replication; electrospray ionization mass spectrometry; noncovalent complex; dis-sociation constant
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