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Multi-wavelength analytical ultracentrifugation as a tool to characterise protein–DNA interactions in solution

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Abstract

Understanding how proteins interact with DNA, and particularly the stoichiometry of a protein–DNA complex, is key information needed to elucidate the biological role of the interaction, e.g. transcriptional regulation. Here, we present an emerging analytical ultracentrifugation method that features multi-wavelength detection to characterise complex mixtures by deconvoluting the spectral signals of the interaction partners into separate sedimentation profiles. The spectral information obtained in this experiment provides direct access to the molar stoichiometry of the interacting system to complement traditional hydrodynamic information. We demonstrate this approach by characterising a multimeric assembly process between the transcriptional repressor of bacterial sialic acid metabolism, NanR and its DNA-binding sequence. The method introduced in this study can be extended to quantitatively analyse any complex interaction in solution, providing the interaction partners have different optical properties.

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Acknowledgements

We are grateful to the New Zealand Royal Society Marsden Fund (RCJD, UOC1506); Ministry of Business, Innovation and Employment Smart Ideas Grant (RCJD, UOCX1706); and the National Institutes of Health Grants (BD, GM120600 and NSF-ACI-1339649). We acknowledge grant support for CRH (Maurice Wilkins Centre). MWL-AUC experiments were performed at the Canadian Center for Hydrodynamics, University of Lethbridge with support from the Canada Foundation for Innovation Grant CFI-37589. UltraScan multi-wavelength development is supported by NIH Grant GM120600 (BD), UltraScan supercomputer calculations were supported through NSF/XSEDE Grant TG-MCB070039N (BD), and University of Texas Grant TG457201 (BD).

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Authors and Affiliations

  1. Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, PO Box 4800, Christchurch, 8140, New Zealand

    Christopher R. Horne & Renwick C. J. Dobson

  2. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, T1K 3M4, Canada

    Amy Henrickson & Borries Demeler

  3. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, 3010, Australia

    Renwick C. J. Dobson

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  1. Christopher R. Horne
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  2. Amy Henrickson
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  3. Borries Demeler
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  4. Renwick C. J. Dobson
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Contributions

CRH designed and performed experiments, and analysed data with support in data interpretation from AH and BD. RCJD supervised the project and co-wrote the paper with CRH. All authors commented on the manuscript.

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Correspondence to Renwick C. J. Dobson.

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Special Issue: Analytical Ultracentrifugation 2019.

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Supplementary Figure 1 Purification of E. coli NanR.

A typical chromatogram from size-exclusion chromatography, where a single monodisperse peak can be observed. Inset. SDS-PAGE analysis showing the increasing purity following each purification step. Lane 1, Protein ladder (kDa); lane 2, crude lysate; lane 3, pooled fractions from anion-exchange chromatography; lane 4, pooled fractions from heparin chromatography; and lane 5, pooled fractions following size-exclusion chromatography (EPS 1953 kb)

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Horne, C.R., Henrickson, A., Demeler, B. et al. Multi-wavelength analytical ultracentrifugation as a tool to characterise protein–DNA interactions in solution. Eur Biophys J 49, 819–827 (2020). https://doi.org/10.1007/s00249-020-01481-6

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  • DOI: https://doi.org/10.1007/s00249-020-01481-6

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