Microsolvation of Charged Sites In Vacuo: Are Native Protein Structures Retained When Charge-Backbone Interactions are Suppressed?

Native mass spectrometry (nMS) is increasingly used to study proteins and their complexes, providing insights into their stoichiometry, topology, and binding affinities. While noncovalent interactions are thought to largely remain intact after desolvation, protein conformation is highly charge-dependent. Increased Coulomb repulsion typically promotes unfolding and charged surface residues engage in interactions with the protein backbone, disrupting hydrogen bonds and distorting secondary structures. However, the relative contributions of these factors to protein unfolding are not well understood. This study investigates how microsolvation of charged sites using crown ethers affects native-like α-helical structures in the gas phase. Using gas-phase fluorescence spectroscopy and ion mobility-mass spectrometry (IM-MS), we find that crown ethers that bind to lysine side chains promote more compact helical conformations, although the charge state still dictates overall compaction. Crown ether variants with different cavity sizes and electron-rich groups showed similar effects, indicating effective occupation of ammonium cations via hydrogen bonding without attenuating charge-charge interactions. These results suggest that while microsolvation can prevent interactions between charged sites and the protein backbone, it has minimal impact on the overall structure compared to Coulomb repulsion. Comparison with solution-phase data reveals significant helical stretching in the gas phase despite microsolvation, further emphasizing the role of Coulomb repulsion in determining biomolecular structure. This work highlights the value of gas-phase fluorescence spectroscopy as a complementary technique to IM-MS for detecting subtle structural changes.