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., and Schaeffer, Patrick M. (2013) Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus. Molecular BioSystems, 9 (12). pp. 3146-3154.

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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.

Item ID: 30493
Item Type: Article (Refereed Research - C1)
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This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

ISSN: 1742-2051
Funders: Smart Futures Fund, National and International Research Alliances Program (NIRAP)
Date Deposited: 04 Dec 2013 09:30
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060101 Analytical Biochemistry @ 100%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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