A1 Refereed original research article in a scientific journal
Sensitive Label-Free Thermal Stability Assay for Protein Denaturation and Protein-Ligand Interaction Studies
Authors: Emmiliisa Vuorinen, Salla Valtonen, Ville Eskonen, Taru Kariniemi, Jelena Jakovleva, Kari Kopra, Harri Härmä
Publisher: AMER CHEMICAL SOC
Publication year: 2020
Journal: Analytical Chemistry
Journal name in source: ANALYTICAL CHEMISTRY
Journal acronym: ANAL CHEM
Volume: 92
Issue: 5
First page : 3512
Last page: 3516
Number of pages: 5
ISSN: 0003-2700
eISSN: 1520-6882
DOI: https://doi.org/10.1021/acs.analchem.9b05712
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/46489723
In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules, as the existing methods utilizing luminescent external probes suffer from low sensitivity. Currently available label-free technologies, e.g., thermal shift assays, circular dichroism, and differential scanning calorimetry, enable studies on protein unfolding and protein-ligand interactions (PLI). Unfortunately, the required micromolar protein concentration increases the costs and predisposes these methods for spontaneous protein aggregation. Here, we report a time-resolved luminescence method for protein unfolding and PLI detection with nanomolar sensitivity. The Protein-Probe method is based on highly luminescent europium chelate-conjugated probe, which is the key component in sensing the hydrophobic regions exposed to solution after protein unfolding. With the same Eu-probe, we also demonstrate ligand-interaction induced thermal stabilization with model proteins. The developed Protein-Probe method provides a sensitive approach overcoming the problems of the current label-free methodologies.
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