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

Abstract

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 D 2 O 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 D 2 O are more stable, with 2−4 K higher melting temperatures than unlabeled proteins in H 2 O. 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 D 2 O 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 D 2 O 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 D 2 O 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 D 2 O. 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.

D2O as an Imperfect Replacement for H2O: Problem or Opportunity for Protein Research?

“…Since contacts between water and protein can reduce the free energy barrier between the different protein conformations, the lower number of water–protein interactions in D 2 O will lead to structurally more stable and less fluctuating proteins, as reported in the literature ( Table 2 ). This proposed stabilization mechanism is also suggested in a recent study by Haidar et al 53 From collision-induced unfolding and ion-mobility mass spectrometry, it was found that the stability of lysozyme, cytochrome c, and bovine ubiquitin in the gas phase is independent of whether the protein is hydrogenated or fully deuterated, in contrast with the increased stability of these proteins in D 2 O solution, again indicating that the changes in protein properties are due to solvent effects. This idea seems to be further confirmed by the general absence of significant differences between the crystal structures of hydrogenated and perdeuterated proteins.…”