Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Abstract

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 × 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 Յ2 × 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.

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

“…33 Thus, since electrostatic interactions are sensitive to the ionic strength of the medium, 34,35 experiments were carried out at different ammonium bicarbonate concentrations ( Figure 5). …”

NanoESI Mass Spectrometry of Rubisco and Rubisco Activase Structures and Their Interactions with Nucleotides and Sugar Phosphates

“…Nevertheless, when nucleotides were added to RA samples that had not been subjected to the dialysis treatment, the binding of a second nucleotide was also observed in abundances similar to that for RA that had been dialysed to remove endogenous ADP. Collisioninduced dissociation (CID) experiments have been used previously in ESI-MS studies of noncovalent complexes to investigate the stability of the interaction [45,46]. A study of the interaction between the Tus replication termination protein and the specific DNA-binding sequence, Ter, examined its dissociation under high salt (800 mM NH 4 OAc) conditions.…”

A eole for the MS analysis of nucleic acids in the post-genomics age

“…Unlike these techniques, MS is capable of resolving any free/bound species at equilibrium in solution, even when such species possess very similar spectroscopic characteristics. With proper experimental design and data treatment, their respective signal intensities can be employed to obtain relative [121,122] and absolute [123][124][125][126][127][128] dissociation constants (K d 's) in solution, matching those afforded by established methods [129]. In this direction, competitive binding experiments in which multiple ligands are mixed simultaneously with the substrate of interest have proven very effective in providing relative scales of binding affinities based on the aspect ratio and distribution of the detected complexes [130 -133].…”