A1 Refereed original research article in a scientific journal
Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability
Authors: Kopra Kari, Valtonen Salla, Mahran Randa, Kapp Jonas N, Hassan Nazia, Gillette William, Dennis Bryce, Li Lianbo, Westover Kenneth D, Plückthun Andreas, Härmä Harri
Publisher: MDPI
Publication year: 2022
Journal: International Journal of Molecular Sciences
Journal name in source: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Journal acronym: INT J MOL SCI
Article number: 7095
Volume: 23
Issue: 13
Number of pages: 16
DOI: https://doi.org/10.3390/ijms23137095
Web address : https://www.mdpi.com/1422-0067/23/13/7095
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/175932873
Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.
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