Generation of a calmodulin-based EPR calcium indicator

Calmodulin is a ubiquitous calcium dependent protein. In the presence of calcium, calmodulin adopts an altered conformation that leads to the generation of downstream cellular calcium signals. Here we describe the introduction of nitroxide EPR probes into calmodulin by means of site-directed spin labeling. These probes sense the calcium-dependent conformational change of calmodulin and, therefore, can serve as calcium indicators. In combination with a light-sensitive calcium chelator, spin-labeled calmodulin can be used to demonstrate calcium release by flash photolysis. These results provide an important step toward describing the molecular dynamics of calcium-induced conformational changes in proteins using EPR spectroscopy.