Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA Kapur, Amit; Beck, Jennifer; Brown, Susan Elizabeth; ...

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Kapur, Amit ; Beck, Jennifer ; Brown, Susan Elizabeth ; Dixon, Nicholas ; Sheil, Margaret
Keywords: ammonium acetate; DNA; mutant protein; regulator protein; Tus protein; unclassified drug; article; binding affinity; dissociation constant; DNA replication; DNA sequence; mass spectrometry; pH; priority journal; protein DNA interaction; Acetates; Base Seq Dissociation constant; DNA replication; Electrospray ionization mass spectrometry; Noncovalent complex; Tus; Social and Behavioral Sciences; Medicine and Health Sciences
Kapur, A., Beck, J. L., Brown, S. E., Dixon, N. E. & Sheil, M. (2002). Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA. Protein Science, 11 (1), 147-157. Tus protein binds tightly to specific DNA sequences (<em>Ter</em>) on the <em>Escherichia coli</em> chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus–<em>Ter</em> 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–<em>Ter</em> complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus–<em>Ter</em> 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 <em>Ter</em> 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 × 10<sup>−9</sup> M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to <em>Ter</em> DNA was estimated, compared with a value of ≤2 × 10<sup>−9</sup> 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 (<em>TerH</em>) that had been identified by database searching.
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