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

Moreau, Morgane J.J.;Schaeffer, Patrick M.

Abstract

The analysis of the salt dependence of protein-DNA complexes provides useful information about the nonspecific 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.

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Molecular BioSystems

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1742-2051

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Royal Society of Chemistry

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DOI

10.1039/c3mb70426b