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Related Experiment Videos

FoldAffinity: binding affinities from nDSF experiments.

Stephan Niebling1,2, Osvaldo Burastero3,4,5, Jérôme Bürgi3

  • 1European Molecular Biology Laboratory - Hamburg Outstation, Notkestr. 85, Hamburg, 22607, Germany. stephan.niebling@embl-hamburg.de.

Scientific Reports
|May 6, 2021
PubMed
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This summary is machine-generated.

This study shows that isothermal analysis of thermal shifts in native differential scanning fluorimetry (nDSF) provides more accurate protein-ligand binding affinities than melting temperatures. A new web server aids accessible application of this method.

Area of Science:

  • Biophysics
  • Biochemistry
  • Structural Biology

Background:

  • Native differential scanning fluorimetry (nDSF) measures protein thermal stability.
  • Ligand binding often increases protein thermal stability, detectable as a shift in nDSF.
  • Quantifying binding affinity from nDSF thermal shifts requires robust analysis.

Purpose of the Study:

  • To evaluate the accuracy of protein-ligand binding affinity quantification using thermal shifts from nDSF experiments.
  • To compare binding affinities derived from isothermal analysis versus melting temperatures.
  • To introduce a web server for accessible isothermal analysis of nDSF data.

Main Methods:

  • Utilized nDSF to study four protein systems with varying ligand binding affinities (nM to high μM).

Related Experiment Videos

  • Performed isothermal analysis of nDSF data to determine binding affinities.
  • Compared nDSF-derived affinities with those from isothermal titration calorimetry (ITC) and microscale thermophoresis (MST).
  • Developed a method for fitting the change in heat capacity upon unfolding during isothermal analysis.

    • Main Results:

    • Binding affinities determined by isothermal analysis showed better agreement with ITC and MST than apparent Kds from melting temperatures.
    • Melting temperature shifts in nDSF may not directly correlate with binding affinity across different systems.
    • The developed isothermal analysis method provides a more reliable quantification of binding affinity.

    Conclusions:

    • Isothermal analysis of nDSF data is a valuable method for accurate protein-ligand binding affinity determination.
    • This approach offers an alternative or complementary method to established techniques like ITC and MST.
    • The released web server facilitates broader adoption of isothermal analysis for nDSF data.