Larson Grant, Bhagi-Damodaran Ambika, Rama Damodaran Anoop
Department of Chemistry, University of Minnesota, Twin Cities, Minneapolis, MN-55455, USA.
bioRxiv. 2025 May 30:2025.05.28.656737. doi: 10.1101/2025.05.28.656737.
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-P]-ATP radiolabeling has long been a conventional method for kinase activity measurements, its reliance on P 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 and hinder scalability. 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 suffer from interference due to intrinsic heme fluorescence and luminescence. In this work, we address this interference by employing a near-IR fluorophore-labeled secondary antibody. Combined with an ATPγS immunoblot-based method, we demonstrate semi-quantitative detection of thiophosphorylation activities of DosS (a prototypical heme-based HK) in different ligation states. Consistent with previous radiolabeling studies, we show DosS's ability for auto-thiophosphorylation is significantly enhanced with CO as compared to O. This low-cost, easy-to-implement method simplifies and democratizes heme-based HK activity measurement.
组氨酸激酶（HKs）是细菌中至关重要的信号转导蛋白，因其关键的细胞功能而成为有吸引力的药物靶点。直接测量其自身磷酸化活性对于开发抑制剂和推进疾病治疗至关重要。虽然[γ-P]-ATP放射性标记长期以来一直是激酶活性测量的传统方法，但其对磷的依赖带来了固有的局限性。该同位素的短半衰期对实验施加了时间敏感的限制，严格的辐射安全和合规要求显著增加了运营成本并阻碍了可扩展性。已经为HKs开发了几种替代方法，包括荧光磷酸结合探针以及基于荧光或发光的抗体，这些方法克服了这些挑战。然而，对于基于血红素的HKs，这些替代方法由于内在的血红素荧光和发光而受到干扰。在这项工作中，我们通过使用近红外荧光团标记的二抗来解决这种干扰。结合基于ATPγS免疫印迹的方法，我们展示了在不同连接状态下对DosS（一种典型的基于血红素的HK）的硫代磷酸化活性进行半定量检测。与先前的放射性标记研究一致，我们表明与氧气相比，一氧化碳存在时DosS的自身硫代磷酸化能力显著增强。这种低成本、易于实施的方法简化并普及了基于血红素的HK活性测量。
