Development of the Fluorescent Biosensor hCalmodulin (hCaM)L39C-monobromobimane(mBBr)/V91C-mBBr, a Novel Tool for Discovering New Calmodulin Inhibitors and Detecting Calcium

A novel, sensible, and specific fluorescent biosensor of human calmodulin (hCaM), namely hCaM L39C-mBBr/V91C-mBBr, was constructed. The biosensor was useful for detecting ligands with opposing fluorescent signals, calcium ions (Ca²⁺) and CaM inhibitors in solution. Thus, the device was successfully applied to analyze the allosteric effect of Ca²⁺ on trifluoroperazine (TFP) binding to CaM (Ca²⁺ K_d = 0.24 μM ± 0.03 with a stoichiometry 4.10 ± 0.15; TFP K_d ∼ 5.74–0.53 μM depending on the degree of saturation of Ca²⁺, with a stoichiometry of 2:1). In addition, it was suitable for discovering additional xanthones (5, 6, and 8) with anti-CaM properties from the fungus Emericella 25379. The affinity of 1–5, 7, and 8 for the complex (Ca²⁺)₄-CaM was excellent because their experimental K_ds were in the nM range (4–498 nM). Docking analysis predicted that 1–8 bind to CaM at sites I, III, and IV as does TFP.