Document Detail

Impact of protein and ligand impurities on ITC-derived protein-ligand thermodynamics.
MedLine Citation:
PMID:  24792574     Owner:  NLM     Status:  Publisher    
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 as significant.
GENERAL SIGNIFICANCE:: Our results indicate that the 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.
Stefan Grüner; Manuel Neeb; Luzi Jakob Barandun; Frank Sielaff; Christoph Hohn; Shun Kojima; Torsten Steinmetzer; François Diederich; Gerhard Klebe
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2014-5-1
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  -     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  2014 May 
Date Detail:
Created Date:  2014-5-5     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2014. Published by Elsevier B.V.
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