Immunoblot-based activity assay for heme-containing histidine kinases

Area of Science:

• Bacterial signal transduction
• Enzyme kinetics
• Drug discovery

Background:

• Histidine kinases (HKs) are vital bacterial proteins regulating cellular processes and are key drug targets.
• Traditional methods for measuring HK autophosphorylation, like radiolabeling with [γ-³²P]-ATP, are limited by isotope half-life, cost, and safety concerns.
• Existing non-radioactive methods often fail for heme-based HKs due to interference from heme's intrinsic fluorescence.

Purpose of the Study:

• To develop a novel, cost-effective, and scalable method for measuring heme-based histidine kinase activity.
• To overcome the limitations of existing assays, particularly interference from intrinsic heme fluorescence.
• To enable semi-quantitative detection of autophosphorylation in heme-based HKs like Mycobacterium tuberculosis DosS.

Main Methods:

• Utilized an ATPγS immunoblot-based assay.
• Employed a near-infrared (near-IR) fluorophore-labeled secondary antibody to detect thiophosphorylation.
• Investigated the activity of Mycobacterium tuberculosis DosS in different ligation states (CO vs. O₂).

Main Results:

• Successfully demonstrated semi-quantitative detection of DosS auto-thiophosphorylation activity.
• Observed significantly enhanced DosS auto-thiophosphorylation in the presence of carbon monoxide (CO) compared to oxygen (O₂).
• Validated the new method's consistency with previous radiolabeling study findings.

Conclusions:

• The developed near-IR antibody and ATPγS immunoblot method effectively measures heme-based HK activity, overcoming intrinsic heme interference.
• This simplified, low-cost assay democratizes the measurement of heme-based HK activity, facilitating drug development.
• The findings provide insights into the regulatory mechanisms of heme-based HKs like DosS.