Hydrophobic peptides affect binding of calmodulin and Ca²⁺ as explored by H/D amide exchange and mass spectrometry

Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca ²⁺ and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high-resolution PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca²⁺, the extent of H/DX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca²⁺-saturated calmodulin exhibits increased protection when bound to the peptides, forming high-affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca²⁺. Titration of the protein using PLIMSTEX provides the Ca²⁺-to-calmodulin binding affinity, which agrees well with previously reported values. The affinities of calmodulin to Ca²⁺ increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The resulting titration data show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca ²⁺-binding properties of this well-studied protein.