A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä

Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions




TekijätMahran Randa, Vello Niklas, Komulainen Anita, Malakoutikhah Morteza, Härmä Harri, Kopra Kari

KustantajaNature Research

Julkaisuvuosi2023

JournalScientific Reports

Tietokannassa oleva lehden nimiScientific Reports

Lehden akronyymiSci. Rep.

Artikkelin numero20066

Vuosikerta13

Numero1

ISSN2045-2322

eISSN2045-2322

DOIhttps://doi.org/10.1038/s41598-023-46720-w(external)

Verkko-osoitehttps://www.nature.com/articles/s41598-023-46720-w(external)

Rinnakkaistallenteen osoitehttps://research.utu.fi/converis/portal/detail/Publication/182193406(external)


Tiivistelmä

Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRASG12C with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRASG12C to those reported by nucleotide exchange assay.


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Last updated on 2025-27-03 at 22:00