Journal article
Impact of protein and ligand impurities on ITC-derived protein-ligand thermodynamics.
- Grüner S Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
- Neeb M Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
- Barandun LJ Laboratorium für Organische Chemie, ETH Zürich, Hönggerberg, HCI, 8093 Zurich, Switzerland.
- Sielaff F Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
- Hohn C Laboratorium für Organische Chemie, ETH Zürich, Hönggerberg, HCI, 8093 Zurich, Switzerland.
- Kojima S Laboratorium für Organische Chemie, ETH Zürich, Hönggerberg, HCI, 8093 Zurich, Switzerland.
- Steinmetzer T Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
- Diederich F Laboratorium für Organische Chemie, ETH Zürich, Hönggerberg, HCI, 8093 Zurich, Switzerland.
- Klebe G Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg, Germany. Electronic address: klebe@staff.uni-marburg.de.
- 2014-05-06
Published in:
- Biochimica et biophysica acta. - 2014
Dynamic light scattering
Enthalpy
Gibbs free energy
Impurities
Isothermal titration calorimetry
Animals
Calorimetry
Cattle
Ligands
Pentosyltransferases
Thermodynamics
Trypsin
English
BACKGROUND
The thermodynamic characterization of protein-ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations.
METHODS
Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC. Data curation was attempted also considering information from analytical techniques to correct stoichiometry.
RESULTS AND CONCLUSIONS
We used trypsin and tRNA-guanine transglycosylase (TGT), together with high affinity ligands to investigate the effect of errors in protein concentration as well as the impact of ligand impurities on the apparent thermodynamics. We found that errors in protein concentration did not change the thermodynamic properties obtained significantly. However, most ligand impurities led to pronounced changes in binding enthalpy. If protein binding of the respective impurity is not expected, the actual ligand concentration was corrected for and the thus revised data compared to thermodynamic properties obtained with the respective pure ligand. Even in these cases, we observed differences in binding enthalpy of about 4kJ⋅mol(-1), which is considered significant.
GENERAL SIGNIFICANCE
Our results indicate that ligand purity is the critical parameter to monitor if accurate thermodynamic data of a protein-ligand complex are to be recorded. Furthermore, artificially changing fitting parameters to obtain a sound interaction stoichiometry in the presence of uncharacterized ligand impurities may lead to thermodynamic parameters significantly deviating from the accurate thermodynamic signature.
The thermodynamic characterization of protein-ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations.
METHODS
Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC. Data curation was attempted also considering information from analytical techniques to correct stoichiometry.
RESULTS AND CONCLUSIONS
We used trypsin and tRNA-guanine transglycosylase (TGT), together with high affinity ligands to investigate the effect of errors in protein concentration as well as the impact of ligand impurities on the apparent thermodynamics. We found that errors in protein concentration did not change the thermodynamic properties obtained significantly. However, most ligand impurities led to pronounced changes in binding enthalpy. If protein binding of the respective impurity is not expected, the actual ligand concentration was corrected for and the thus revised data compared to thermodynamic properties obtained with the respective pure ligand. Even in these cases, we observed differences in binding enthalpy of about 4kJ⋅mol(-1), which is considered significant.
GENERAL SIGNIFICANCE
Our results indicate that ligand purity is the critical parameter to monitor if accurate thermodynamic data of a protein-ligand complex are to be recorded. Furthermore, artificially changing fitting parameters to obtain a sound interaction stoichiometry in the presence of uncharacterized ligand impurities may lead to thermodynamic parameters significantly deviating from the accurate thermodynamic signature.
- Language
-
- English
- Open access status
- closed
- Identifiers
-
- DOI 10.1016/j.bbagen.2014.04.018
- PMID 24792574
- Persistent URL
- https://folia.unifr.ch/global/documents/223252
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