TY - JOUR
T1 - Multi-wavelength analytical ultracentrifugation as a tool to characterise protein–DNA interactions in solution
AU - Horne, Christopher R.
AU - Henrickson, Amy
AU - Demeler, Borries
AU - Dobson, Renwick C.J.
N1 - Publisher Copyright:
© 2020, European Biophysical Societies' Association.
PY - 2020/12
Y1 - 2020/12
N2 - 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.
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.
KW - Multi-wavelength analytical ultracentrifugation
KW - NanR
KW - Protein–DNA interaction
KW - Sedimentation velocity
KW - Sialic acid
UR - http://www.scopus.com/inward/record.url?scp=85096292109&partnerID=8YFLogxK
U2 - 10.1007/s00249-020-01481-6
DO - 10.1007/s00249-020-01481-6
M3 - Article
C2 - 33219833
AN - SCOPUS:85096292109
SN - 0175-7571
VL - 49
SP - 819
EP - 827
JO - European Biophysics Journal
JF - European Biophysics Journal
IS - 8
ER -