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

doi:10.1007/s00249-020-01481-6

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

Chemistry and Biochemistry

Research output: Contribution to journal › Article › peer-review

11 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 language	English
Pages (from-to)	819-827
Number of pages	9
Journal	European Biophysics Journal
Volume	49
Issue number	8
DOIs	https://doi.org/10.1007/s00249-020-01481-6
State	Published - Dec 2020

Funding

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). 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).

Funders	Funder number
UOC1506
TG457201
TG-MCB070039N
NSF-ACI-1339649, GM120600
Canada Foundation for Innovation	CFI-37589
Canada Foundation for Innovation
Ministry of Business, Innovation and Employment	UOCX1706

Keywords

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

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10.1007/s00249-020-01481-6

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

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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AU - Demeler, Borries

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PY - 2020/12

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AB - 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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KW - Sedimentation velocity

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

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