Analysis of Calmodulin-Melittin Interactions Through Hydrogen-Deuterium Exchange and Mass Spectrometry

Calmodulin is a ubiquitous, highly conserved regulatory protein which binds a variety of small peptides with high affinity when in the presence of Ca²⁺. The calmodulin system can serve as a model for other protein-ligand interactions due to the drastic conformational change that calmodulin undergoes when binding peptide ligands. One such peptide is melittin, the primary constituent of honeybee venom. The calmodulin-melittin system is not fully characterized and a crystal structure for it is not available, though it is presumed to be similar to the complexes formed by other peptides binding to calmodulin. In this study, we investigate the locations on both melittin and calmodulin where the binding interaction takes place. We use hydrogen-deuterium exchange (H/DX) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Collision induced dissociation (CID) and protein digestion allow us to achieve structural resolution at the peptide level. During initial deuteration experiments, we saw an increase in mass in both calmodulin and melittin, which is indicative of H/DX occurring. A decrease in deuterium uptake due to an increase in protection of the exchangeable hydrogens in the interacting protein and ligand has been observed. Therefore, the combination of H/DX with CID and protein digestion is an effective method for identifying interacting fragments in this system. We anticipate observing binding and protection in the last twelve residues of melittin, due to similarities with other, better characterized, ligands of calmodulin. This study should lead to a greater understanding of the calmodulin-melittin system and the nature of their interaction.