The sheet-to-helix transition is a potential gas-phase unfolding pathway for a multidomain protein CRM.

Despite the expansive applications of gas-phase unfolding techniques, the molecular mechanism for the solvent-free forced unfolding pathway which substrate multidomain proteins usually adopt remains elusive at the secondary structure level. Herein, upon carefully selecting CRM as a therapeutically-relevant model system containing multiple secondary structure-separated domains, we systematically examine its solvent-free unfolding pathway. Further-more, utilizing the hybrid of noncovalent chemical probing with niacinamide and ion mobility-mass spectrometry-guided all-atom molecular dynamics simulations, we map a nearly complete unfolding atlas for the conjugate vaccine carrier protein CRM in a domain- and secondary structure-resolved manner. The totality of our data supports the preferential unfolding of the sheet-rich domain, indicating the dynamic transition from β-sheet to α-helices, and demonstrating that helices exhibit comparatively higher stability than β-sheets. We propose that this sheet-to-helix dynamic transition may be central to the gas-phase unfolding pathways of multidomain proteins, suggesting the need for systematic studies on additional multidomain protein systems.