@article{8a1dbf71b69c4c24bd948eb8f9722e93,
title = "Complementary protocols to evaluate inhibitors against the DnaK chaperone network",
abstract = "Bacterial DnaK belongs to the Hsp70 chaperone family, which plays a critical role in maintaining proteostasis by catalyzing protein folding, and is a proposed antibacterial target in the pathogen Mycobacterium tuberculosis. Here, we describe an experimental toolbox for evaluating inhibitors against the mycobacterial DnaK chaperone network: a coupled-enzymatic assay to monitor ATPase activity, a proteolytic cleavage assay to study DnaK conformational changes upon ligand addition, as well as a protein renaturation assay to assess chaperone function. For complete details on the use and execution of this protocol, please refer to Hosfelt et al. (2021).",
keywords = "Molecular/Chemical Probes, Protein Biochemistry",
author = "Aweon Richards and Yawson, {Gideon K.} and Brock Nelson and Lupoli, {Tania J.}",
note = "Funding Information: The authors would like to thank New York University (FAS) for funding. We would like to acknowledge Jordan Hosfelt for initial testing of some methods. pET His6 Sumo TEV LIC cloning vector (2S-T) was a gift from Scott Gradia (Addgene plasmid # 29711; http://n2t.net/addgene:29711; RRID: Addgene_29711). pHYRS52 was a gift from Hideo Iwai (Addgene plasmid # 31122; http://n2t.net/addgene:31122; RRID: Addgene_31122). Conception and design, T.L. A.R. G.Y. and B.N.; investigation, A.R. G.Y. and B.N.; analysis and interpretation of data, T.L. A.R. G.Y. and B.N.; writing of the manuscript, A.R. T.L. G.Y. and B.N.; administrative, technical, or material support, T.L. The authors declare no competing interests. Funding Information: The authors would like to thank New York University (FAS) for funding. We would like to acknowledge Jordan Hosfelt for initial testing of some methods. pET His6 Sumo TEV LIC cloning vector (2S-T) was a gift from Scott Gradia (Addgene plasmid # 29711; http://n2t.net/addgene:29711 ; RRID: Addgene_29711). pHYRS52 was a gift from Hideo Iwai (Addgene plasmid # 31122; http://n2t.net/addgene:31122 ; RRID: Addgene_31122). Publisher Copyright: {\textcopyright} 2022 The Author(s)",
year = "2022",
month = jun,
day = "17",
doi = "10.1016/j.xpro.2022.101381",
language = "English (US)",
volume = "3",
journal = "STAR Protocols",
issn = "2666-1667",
publisher = "Cell Press",
number = "2",
}