Hydrophobic Shielding Preserves Transmembrane Secondary Structure in the Gas Phase.

Electrospray ionization mass spectrometry (ESI-MS) can transfer membrane proteins into the gas phase under native-like conditions, preserving noncovalent assemblies and overall folds. Nevertheless, how much of their secondary structure survives after desolvation remains elusive. Here we address this issue using long-time-scale (submillisecond) molecular dynamics (MD) simulations to study how membrane proteins preserve architecture in the gas phase and examine residue-level determinants of transmembrane (TM) stability. We selected four representative membrane proteins with distinct oligomeric states and TM topologies to capture β-barrel, helix bundle, monomeric G-protein-coupled receptor, and multimeric mechanosensitive channel archetypes. Although local unfolding and conformational rearrangements occur in the extramembrane and intramembrane regions, the TM domains remain stable. This stability arises from the preorganized hydrophobic environment of the membrane, which shapes the TM architecture into a configuration inherently compatible with the apolar gas phase. Furthermore, the vacuum environment reinforces hydrophobic packing, allowing the TM secondary structure to remain intact. Together, these factors highlight the protective role of surface-exposed hydrophobic residues in maintaining the TM secondary structure. Our results identify hydrophobic surface area and oligomeric interfaces as primary protectants of TM secondary structure under ESI-MS conditions and establish a mechanistic framework for understanding membrane protein stability in the absence of lipids.