Development of new hCaM-Alexa Fluor^® biosensors for a wide range of ligands

Eight new fluorescent biosensors of human calmodulin (hCaM) using Alexa Fluor^® 350, 488, 532, and 555 dyes were constructed. These biosensors are thermodynamically stable, functional, and highly sensitive to ligands of the CaM. They resolve the problem of CaM ligands with similar spectroscopic properties to the intrinsic and extrinsic fluorophores of other biosensors previously reported. Additionally, they can be used in studies of protein-protein interaction through Förster resonance energy transfer (FRET). The variation in T_m (range 78.07-81.47 °C; 79.05 to WT) is no larger than two degrees in all cases in regards to CaM WT. The K_ds calculated with all biosensors for CPZ and BIMI (a new inhibitor of CaM) are in the range of 0.45-1.86 and 0.69-1.54 μm respectively. All biosensors retain their ability to activate Calcineurin about 70%. Structural models built "in silico" show their possible conformation taking the fluorophores in protein thus we can predict system stability. Finally, these new biosensors represent a biotechnological development applied to an analytical problem, which aims to determine accurately the affinity of inhibitors of CaM without possible interference, to be put forward as possible drugs related to CaM.