Inter-Domain Repulsion of Dumbbell-Shaped Calmodulin during Electrospray Ionization Revealed by Molecular Dynamics Simulations

The mechanisms whereby protein ions are released fromnanodropletsat the liquid-gas interface have continued to be controversialsince electrospray ionization (ESI) mass spectrometry was widely appliedin biomolecular structure analysis in solution. Several viable pathwayshave been proposed and verified for single-domain proteins. However,the ESI mechanism of multi-domain proteins with more complicated andflexible structures remains unclear. Herein, dumbbell-shaped calmodulinwas chosen as a multi-domain protein model to perform molecular dynamicssimulations to investigate the structural evolution during the ESIprocess. For [Ca(4)CAM], the protein followed the classicalcharge residue model. As the inter-domain electrostatic repulsionincreased, the droplet was found to split into two sub-droplets, whilestronger-repulsive apo-calmodulin unfolded during the early evaporationstage. We designated this novel ESI mechanism as the domain repulsionmodel, which provides new mechanistic insights into further explorationof proteins containing more domains. Our results suggest that greaterattention should be paid to the effect of domain-domain interactionson structure retention during liquid-gas interface transferwhen mass spectrometry is used as the developing technique in gasphase structural biology.

Automated summary

In this study, the dumbbell-shaped calmodulin was chosen as a multi-domain protein model to investigate the structural evolution during electrospray ionization (ESI) process. The results showed that the protein followed the classical charge residue model and the apo-calmodulin underwent unfolding during the early evaporation stage. This study proposed a novel ESI mechanism called the domain repulsion model, which provides new mechanistic insights for the exploration of proteins with more domains.