Probing Conformational and Functional Substates of Calmodulin by High-Pressure FTIR

Abstract

In response to intracellular Ca2+-concentration changes, the highly dynamic and flexible 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. K-Ras4B, known as a key regulator in the ERK pathway, was identified as specific binding partner of CaM, causing the dissociation of the lipidated K-Ras4B from the plasma membrane and thus reducing its activity. However, the interaction of K-Ras4B and CaM has not been elucidated in detail until now. In order to evaluate the conformational space of CaM and shed more light on the mechanism of subsequent target recognition and protein function, identification and characterization of functionally relevant conformational substates is mandatory. Applying pressure in combination with spectroscopies such as FTIR spectroscopy enables to populate and probe otherwise transient low-lying excited conformational substates of CaM close in energy to ground state, but with a smaller partial volume. Evidence has grown that these states are functionally relevant, for instance 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 C-terminal K-Ras4B peptide. We show that not only Ca2+-binding, but also the presence of the target peptide has a drastic effect on the conformational dynamics of the protein.