A DNA-based nanomechanical device used to characterize the distortion of DNA by apo-SoxR protein

Chunhua Liu, Eunsuk Kim, Bruce Demple, Nadrian Seeman

Research output: Contribution to journalArticle

Abstract

DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple-crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In our case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to ∼2 bp. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding of the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol.

Original languageEnglish (US)
Pages (from-to)937-943
Number of pages7
JournalBiochemistry
Volume51
Issue number5
DOIs
StatePublished - Feb 7 2012

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Equipment and Supplies
DNA
Fluorescence Resonance Energy Transfer
Proteins
Binding Sites
Free energy
Integration Host Factors
Oxidation-Reduction
Carrier Proteins
Coloring Agents
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

A DNA-based nanomechanical device used to characterize the distortion of DNA by apo-SoxR protein. / Liu, Chunhua; Kim, Eunsuk; Demple, Bruce; Seeman, Nadrian.

In: Biochemistry, Vol. 51, No. 5, 07.02.2012, p. 937-943.

Research output: Contribution to journalArticle

Liu, Chunhua ; Kim, Eunsuk ; Demple, Bruce ; Seeman, Nadrian. / A DNA-based nanomechanical device used to characterize the distortion of DNA by apo-SoxR protein. In: Biochemistry. 2012 ; Vol. 51, No. 5. pp. 937-943.
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