Thermal Shift Assay for Characterizing the Stability of RNA Helicases and Their Interaction with Ligands.

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  • Author(s): Saridakis E;Saridakis E; Coste F; Coste F
  • Source:
    Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2209, pp. 73-85.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE
    • Publication Information:
      Publication: Totowa, NJ : Humana Press
      Original Publication: Clifton, N.J. : Humana Press,
    • Subject Terms:
      Bacterial Proteins/*chemistry ; Fluorometry/*methods ; RNA Helicases/*chemistry ; Rho Factor/*chemistry; Mycobacterium tuberculosis/enzymology ; Protein Stability ; Temperature
    • Abstract:
      Thermofluor or thermal shift assay is an easily implementable, high-throughput method for assessing the thermostability of proteins and the influence of various ligands on that stability. It is particularly useful for the assaying of ligands that may stabilize oligomeric helicases, which rely on both substrates (oligonucleotides) and nucleotide cofactors (ATP analogues) for their stability in a functional state. In this chapter, we describe the rationale and present a basic protocol for the use of this technique. Multi-ligand screening is also discussed via a worked example of the stabilization of a hexameric RNA helicase, a target protein for structural studies in our laboratories.
    • Contributed Indexing:
      Keywords: Differential scanning fluorimetry; Melting temperature; RNA helicase; Thermal shift assay; Thermofluor; Thermostability
    • Accession Number:
      0 (Bacterial Proteins)
      0 (Rho Factor)
      EC 3.6.4.13 (RNA Helicases)
    • Publication Date:
      Date Created: 20201117 Date Completed: 20210331 Latest Revision: 20210331
    • Publication Date:
      20240829
    • Accession Number:
      10.1007/978-1-0716-0935-4_5
    • Accession Number:
      33201463