Fluorescence polarization assay for calmodulin binding to plasma membrane Ca 2+–ATPase: Dependence on enzyme and Ca 2+ concentrations

Abstract

Calmodulin (CaM) is a Ca 2+ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K d for the interaction of CaM with the plasma membrane Ca 2+–ATPase (PMCA), a Ca 2+ pump regulated by binding of CaM. Previous assays of PMCA–CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca 2+ dependence of CaM binding to PMCA. FP assays directly detect CaM–target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA ( K d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca 2+ dependence follows a simple Hill plot demonstrating cooperative binding of Ca 2+ to the binding sites in CaM.