title: A DNA-based nanomechanical device used to characterize the distortion of DNA by Apo-SoxR protein.
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.
authors.affiliation: Department of Chemistry, New York University, New York, New York 10003, United States.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Liu
authors.forename: Eunsuk
authors.initials: E
authors.lastname: Kim
authors.forename: Bruce
authors.initials: B
authors.lastname: Demple
authors.forename: Nadrian C
authors.initials: NC
authors.lastname: Seeman
mesh_terms: D001059:Apoproteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000295:instrumentation*; D009690:Nucleic Acid Conformation; D014157:Transcription Factors / Q000737:chemistry*
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C070724:SoxR protein, Bacteria