Abstract
Native mass spectrometry is widely used to interrogate protein systems by gently transferring them into the gas phase, where they are thought to persist in a native-like structure. However, which aspects of the solution structure survive solvent removal remains unclear. Protein folding in solution is cooperative, largely due to the solvent-mediated hydrophobic effect, and its disappearance in vacuo is therefore expected to eliminate thermodynamic cooperativity, leading to independent loss of native contacts and structurally heterogeneous ensembles. Here, we determine which features of the canonical protein holo-myoglobin persist in the solvent-free state by delineating its gas-phase denaturation and unfolding pathways. Integrating tandem-trapped ion mobility spectrometry/tandem-mass spectrometry (Tandem-TIMS) with molecular dynamics simulations reveals that native-like holo-myoglobin largely retains its α-helical fold, compact core architecture, and noncovalent heme coordination in the absence of solvent. Energetic activation in vacuo indicates a hierarchical unfolding mechanism: an initial denaturation within a compact shape, followed by global unfolding, and eventual heme loss. Notably, denaturation proceeds collectively across multiple helices and is largely insensitive to salt-bridge rearrangements, arguing against electrostatic interactions as the primary stabilizing factor of native-like protein structures in the absence of solvent. Instead, our findings propose steric and topological constraints as key contributors to the kinetic barriers that confer metastability on solution-like protein structures in vacuo. These findings bear directly on the interpretation of native ion mobility/mass spectrometry, which assumes that kinetically trapped solvent-free protein structures reflect aspects of their solution-phase topology. By clarifying the physical origin of this metastability, our results provide a structural framework for interpreting native mass spectrometry measurements.