Electrospray ionisation mass spectrometry of non-convalent complexes of a DNA-binding protein, TUS with its DNA recognition sequence, TER B

Full text: Electrospray ionization mass spectrometry (ESI MS) is a powerful tool for analysis of biological macromolecules. More recently, the technique has been applied to characterization of non covalent complexes. The application of ESI MS to the study of these biochemical complexes presents a number of technical difficulties. For example traditional buffer components are not volatile and are therefore not compatible with this technique. Observations of macromolecular complexes in the gas phase require careful scrutiny. Are they representative of the solution structure of the complex or are they the result of non-specific gas phase associations of molecules? In order to examine some of these questions, we have studied the complex of Tus protein (35,652 Da), with its double-stranded 21 mer DNA recognition sequence, TerB. Binding of Tus to termination sequences (Ter) on the E coll chromosome halts DNA replication. The Tus Ter B complex is well-characterized, a crystal structure is available, and the binding characteristics of a range of protein mutants and variant DNA sequences have been examined using several techniques. We have determined conditions for detecting a Tus Ter B complex by ESI MS. Under these conditions, only a negligible amount of a Tus random DNA complex was observed. We have also been able to distinguish binding between wild-type and mutant Tus to Ter B, and between Ter B and variant DNA sequences to Tus