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Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

  1. Author:
    Kopra, Kari [ORCID]
    Valtonen, Salla [ORCID]
    Mahran, Randa
    Kapp, Jonas N [ORCID]
    Hassan, Nazia
    Gillette,Bill [ORCID]
    Dennis, Bryce
    Li, Lianbo [ORCID]
    Westover, Kenneth D [ORCID]
    Plückthun, Andreas [ORCID]
    Härmä, Harri

  2. Author Address

    Department of Chemistry, University of Turku, Henrikinkatu 2, 20500 Turku, Finland., Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland., Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, 8560 Progress Dr., Frederick, MD 21702, USA., Departments of Biochemistry and Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, L4.270, Dallas, TX 75390, USA.,
    1. Year: 2022
    2. Date: Jun 26
    3. Epub Date: 2022 06 26
  2. Journal: International Journal of Molecular Sciences
    1. 23
    2. 13
  4. Type of Article: Article
  5. Article Number: 7095
  1. Abstract:

    Thermal unfolding methods are commonly used as a predictive technique by tracking the protein 39;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 39;Gold Standard 39; 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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External Sources

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  2. DOI: 10.3390/ijms23137095
  3. PMID: 35806100
  4. PMCID: PMC9266822
  5. PII : ijms23137095

Library Notes

  1. Fiscal Year: FY2021-2022
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