Effects of Hydrogen/Deuterium Exchange on Protein Stability in Solution and in the Gas Phase

Mass spectrometry (MS)-based techniques are widely used for probing protein structure and dynamics in solution. H/D exchange (HDX)-MS is one of the most common approaches in this context. HDX is often considered to be a "benign" labeling method, in that it does not perturb protein behavior in solution. However, several studies have reported that D₂O pushes unfolding equilibria toward the native state. The origin, and even the existence of this protein stabilization remain controversial. Here we conducted thermal unfolding assays in solution to confirm that deuterated proteins in D₂O are more stable, with 2-4 K higher melting temperatures than unlabeled proteins in H₂O. Previous studies tentatively attributed this phenomenon to strengthened H-bonds after deuteration, an effect that may arise from the lower zero-point vibrational energy of the deuterated species. Specifically, it was proposed that strengthened water-water bonds (W···W) in D₂O lower the solubility of nonpolar side chains. The current work takes a broader view by noting that protein stability in solution also depends on water-protein (W···P) and protein-protein (P···P) H-bonds. To help unravel these contributions, we performed collision-induced unfolding (CIU) experiments on gaseous proteins generated by native electrospray ionization. CIU profiles of deuterated and unlabeled proteins were indistinguishable, implying that P···P contacts are insensitive to deuteration. Thus, protein stabilization in D₂O is attributable to solvent effects, rather than alterations of intraprotein H-bonds. Strengthening of W···W contacts represents one possible explanation, but the stabilizing effect of D₂O can also originate from weakened W···P bonds. Future work will be required to elucidate which of these two scenarios is correct, or if both contribute to protein stabilization in D₂O. In any case, the often-repeated adage that "D-bonds are more stable than H-bonds" does not apply to intramolecular contacts in native proteins.