Dissecting the salt dependence of the Tus–Ter protein–DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus

Abstract

The analysis of the salt dependence of protein–DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic T_m peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus–Ter and Tus–Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus–Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher K_spe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein–DNA complexes.