Protonation State-Induced Unfolding of Protein Secondary Structure in the Gas Phase.

The combination of infrared spectroscopy (IR) and ion mobility mass spectrometry (IM-MS) has revealed that protein secondary structures are retained upon transformation from aqueous solution to the gas phase under gentle conditions. Yet the details about where and how these structural elements are embedded in the gas phase remain elusive. In this study, we employ long time scale molecular dynamics (MD) simulations to examine the extent to which proteins retain their solution structures and the impact of protonation state on the stability of secondary structures in the gas phase. Our investigation focuses on two well-studied proteins, myoglobin and β-lactoglobulin, representing typical helical and β-sheet proteins, respectively. Our simulations accurately reproduce the experimental collision cross section (CCS) data measured by IM-MS. Based on accurately reproducing previous experimental collision cross section data and dominant secondary structural species obtained from IM-MS and IR, we confirm that both proteins largely retain their native secondary structural components upon passing from aqueous solution to the gas phase. However, we observe significant reductions in secondary structure contents (19.2 ± 1.2% for myoglobin and 7.3 ± 0.6% for β-lactoglobulin) in specific regions predominantly composed of ionizable residues. Further mechanistic analysis suggests that alterations in protonation states of these residues after phase transition induce changes in their local interaction networks and backbone dihedral angles, which potentially promote the unfolding of secondary structures in the gas phase. We anticipate that similar protonation state induced unfolding may be observed in other proteins possessing distinct secondary structures. Further studies on a broader array of proteins will be essential to refine our understanding of protein structural behavior during the transition to the gas phase.