Binding affinity quantifications using microscale thermophoresis (MST)

Affinity measurements of purified Mom with various ligands were carried out on a Monolith NT.115 instrument (NanoTemper Technologies GmbH). Mom was fluorescently labeled using NanoTemper's Protein Labeling Kit RED-NHS or RED-tris-NTA dye (for his-tagged Mom and mutants). Ligands including acetyl CoA, S-adenosyl methionine (SAM), coenzyme A (CoASH), malonyl coenzyme A and salts of various metal ions were resuspended or dissolved in MST buffer (10 mM HEPES–NaOH (pH 7.4), 300 mM NaCl and 14 mM β-mercaptoethanol) to prepare a 16 step two-fold dilution series. Diluted ligands were incubated with 50 nM of labeled Mom at room temperature for 5 min and reactions were spun at 17000g for 5 min. Supernatants were loaded into NanoTemper premium coated capillaries. MST measurements were performed at 25°C using 60% MST power and 90% LED power. Data analyses were carried out using NanoTemper analysis software.