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Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions
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Complementary protocols to evaluate inhibitors against the DnaK chaperone network.

Aweon Richards1, Gideon K Yawson1, Brock Nelson1

  • 1Department of Chemistry, New York University, New York, NY 10003, USA.

STAR Protocols
|May 23, 2022
PubMed
Summary

Researchers developed a toolbox to test inhibitors targeting the bacterial DnaK chaperone network, crucial for protein folding and a potential target against Mycobacterium tuberculosis. This toolbox aids in evaluating new antibacterial strategies.

Keywords:
Molecular/Chemical ProbesProtein Biochemistry

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Microbiology

Background:

  • Bacterial DnaK, an Hsp70 chaperone, is vital for maintaining proteostasis by aiding protein folding.
  • DnaK is a validated antibacterial target in Mycobacterium tuberculosis, a significant human pathogen.

Purpose of the Study:

  • To present a comprehensive experimental toolbox for assessing inhibitors of the mycobacterial DnaK chaperone network.
  • To facilitate the evaluation of potential antibacterial compounds targeting DnaK function.

Main Methods:

  • A coupled-enzymatic assay was employed to quantify DnaK's ATPase activity.
  • Proteolytic cleavage assays were utilized to detect ligand-induced conformational changes in DnaK.
  • Protein renaturation assays were performed to evaluate the functional impact of DnaK inhibitors on chaperone activity.

Main Results:

  • The described assays provide a robust framework for inhibitor screening against mycobacterial DnaK.
  • The toolbox allows for the characterization of DnaK inhibitors at enzymatic, conformational, and functional levels.

Conclusions:

  • This experimental toolbox is essential for the development of novel antibacterial agents targeting the DnaK chaperone in Mycobacterium tuberculosis.
  • The described methods enable comprehensive evaluation of DnaK inhibitors, advancing the search for new tuberculosis treatments.