Immunoblot-based activity assay for heme-containing histidine kinases

Histidine kinases (HKs) are essential bacterial signal transduction proteins and attractive drug targets due to their critical cellular functions. Direct measurement of their autophosphorylation activity is crucial for developing inhibitors and advancing disease treatments. While [γ-32P]-ATP radiolabeling has long been a conventional method for kinase activity measurements, its reliance on 32P introduces inherent limitations. The isotope’s short half-life imposes time-sensitive constraints on experiments, and stringent radiation safety and compliance requirements significantly increase operational costs. Several alternative methods including fluorescent phosphate-binding probes and fluorescence- or luminescence-based antibodies have been developed for HKs that overcome these challenges. However, for heme-based HKs, these alternatives are prone to interference from the luminescence of heme or fluorescence from a minor fraction of mismetalated ZnII-bound protoporphyrin IX that is retained on the protein after gel electrophoresis. In this work, we address this interference by employing a near-IR (NIR) fluorophore-labeled secondary antibody and combining it with an ATPγS-utilizing immunoblot-based method to demonstrate ligation dependent thiophosphorylation activities of two heme-based HKs, DosS and GcHK. Consistent with previous studies, our NIR fluorescence-based immunoblot method reveals that GcHK is activated by ligand binding (O2/CO), whereas the O2-sensor DosS is inhibited upon binding O2. Overall, this easy-to-implement method simplifies and democratizes activity measurements of heme-based HKs.