Abstract:
The ubiquitous Ca2+-sensing protein calmodulin (CaM) interacts with more than 300 diverse target proteins that are involved in numerous signaling pathways in eukaryotic cells. This unique promiscuous target binding behavior and the underlying functional versatility of CaM is a result of its structural flexibility. CaM spans multiple conformational substates in solution providing adaptable binding surfaces for different target proteins. The conformational space of this protein needs to be explored to shed more light on the mechanism of target recognition and protein function. Here, we used pressure modulation in combination with FTIR spectroscopy to populate and probe otherwise transient low-lying excited conformational substates of CaM close in energy to its ground state, which are supposed to be functionally relevant in recognition and ligand binding events. The pressure-induced conformational changes of CaM were studied in its Ca2+-free and Ca2+-bound state and in the presence of the hypervariable region (HVR) of the signaling peptide K-Ras4B as a binding partner. We demonstrate that the conformational dynamics of CaM is vastly affected by binding of both Ca2+ ions and the lipidated signaling peptide K-Ras4B. Moreover, we could uncover conformational substates of CaM by pressure perturbation that are partially unfolded and more solvated and conceivably facilitate target recognition by exposing the required binding surfaces.