10 μM of Rv0158 protein (in 50 mM Tris pH 7.5, 100 mM NaCl) was labelled using RED-NHS MonolithTM Protein Labeling Kit (NanoTemper Technologies, cat. no.- MO-L011) according to manufacturer’s instructions. Each of the small molecule ligands (coA (Sigma Aldrich, cat. no.- C4282),
acetyl-CoA (Sigma Aldrich, cat. no.- A2056),
propionyl-CoA (Sigma Aldrich, cat. no.- P5397),
malonyl-CoA (Sigma Aldrich, cat. no.- M4263),
methylmalonyl- CoA (Sigma Aldrich, cat. no.- M1762), C10-CoA (Sigma Aldrich, cat. no.- D5269), C12-CoA (Sigma Aldrich, cat. no.- L2659),
C16-CoA (Sigma Aldrich, cat. no.- P9716), C18-CoA (Sigma Aldrich, cat. no.- S0802)) were titrated against the labeled protein in 1:1 dilution series starting with the highest ligand concentration of of 75 μM. A total of 16 twofold serial dilutions of the target ligand were prepared. The labeled protein was added such that the final concentration of the protein is 5 nM. Experiments were carried out using MonolithTM NT.115 MST Standard Capillaries (NanoTemper Technologies, cat. no.- MO-K022) and measured using a Monolith NT.115 instrument with MO.Control software. Curves were fitted with a single-site binding model using PALMIST 1.5.6 software (Scheuermann et al., 2016 (
link)) and figures were generated using GUSSI software v1.1.0 (Brautigam, 2015 (
link)).
Shee S., Veetil R.T., Mohanraj K., Das M., Malhotra N., Bandopadhyay D., Beig H., Birua S., Niphadkar S., Nagarajan S.N., Sinha V.K., Thakur C., Rajmani R.S., Chandra N., Laxman S., Singh M., Samal A., Seshasayee A.N, & Singh A. (2023). Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis. eLife, 12, e80218.
