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

Calmodulin undergoes significant conformation change in the presence of Ca2+ and hydrophobic peptides. [Display omitted] ▶ Capabilities of MS-based PLIMSTEX illustrated for calmodulin-peptide binding. ▶ Digestion of calmodulin following HDX reveals binding at component peptide level. ▶ The presence of peptide ligands increases Ca2+ binding affinity to calmodulin. ▶ Similar HDX patterns for binding of three peptides suggest common structures. Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca2+ 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 Ca2+-binding properties of this well-studied protein.