Application of a Fluorescent Biosensor in Determining the Binding of 5-HT to Calmodulin

Abstract

Here, we show the utility of the fluorescent biosensor hCaM-M124C-mBBr in detecting and determining the affinity of serotonin (5-HT). We obtained a Kd of 5-HT (0.71 μm) for the first time, the same order of magnitude as most anti-CaM drugs. This data can contribute to understanding the direct and indirect modulation of CaM on its binding proteins when the 5-HT concentration varies in different tissues or explain some of the side effects of anti-CaM drugs. On the other hand, molecular modeling tools help the rational design of biosensors and adequately complement the experimental results. For example, the docking study indicates that 5-HT binds at the same site as chlorpromazine (site 1) with a theoretical Ki of 2.84 μM; while the molecular dynamics simulations indicate a stability of the CaM–5-HT complex with a theoretical ΔG of −4.85 kcal mol−1, where the enthalpy contribution is greater. Thus, the combination of biotechnology and bioinformatics helps in the design and construction of more robust biosensors.