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Issue 12, 2013
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Dissecting the salt dependence of the Tus–Ter protein–DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus

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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 Tm 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 Kspe 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.

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

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Publication details

The article was received on 15 Jul 2013, accepted on 30 Sep 2013 and first published on 01 Oct 2013


Article type: Paper
DOI: 10.1039/C3MB70426B
Citation: Mol. BioSyst., 2013,9, 3146-3154
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    Dissecting the salt dependence of the Tus–Ter protein–DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus

    M. J. J. Moreau and P. M. Schaeffer, Mol. BioSyst., 2013, 9, 3146
    DOI: 10.1039/C3MB70426B

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