Microscale thermophoresis and fluorescence polarization assays of calcineurin-peptide interactions

Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It is very important to study the affinity between calcineurin and its substrate or other interacting proteins. Two conserved motifs have been reported on the interactive proteins of calcineurin, namely, the PxIxIT motif and the LxVP motif. Here, we used 5(6)-carboxyfluorescein to fluorescently label the N-terminus of the short peptides derived from the two motifs and then determined the affinity between the protein and polypeptides. Microscale thermophoresis (MST) is very suitable for determining calcineurin with peptides containing the LxVP motif. The K-d values of the binding of calcineurin with NFATc1-YLAVP, NHE1-YLTVP, and A238L-FLCVK peptides were 6.72 +/- 0.19 mu M, 17.14 +/- 0.35 mu M, and 15.57 +/- 0.10 mu M, respectively. The GST pull-down results further confirmed the binding trend of the three peptides to calcineurin. However, fluorescently labeled PxIxIT polypeptides are not suitable for MST due to their own aggregation. We determined the binding affinity of the RCAN1-PSVVVH polypeptide to calcineurin by the fluorescence polarization (FP) method. MST and FP assays are fast and accurate in determining the affinity between protein-peptide interactions. Our research laid the foundation for screening the molecules that affect the binding between calcineurin and its substrates in the future.

Automated summary

This study determined the affinity between calcineurin and peptides using fluorescent labeling and two conserved motifs, showing that MST and FP assays are fast and accurate in determining protein-peptide interactions. Results laid the foundation for future screening of molecules affecting the binding between calcineurin and its substrates.