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

Christopher R. Horne, Amy Henrickson, Borries Demeler, Renwick C.J. Dobson

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)819-827
Number of pages9
JournalEuropean Biophysics Journal
Volume49
Issue number8
DOIs
StatePublished - Dec 2020
Externally publishedYes

Keywords

  • Multi-wavelength analytical ultracentrifugation
  • NanR
  • Protein–DNA interaction
  • Sedimentation velocity
  • Sialic acid

ASJC Scopus subject areas

  • Biophysics

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