Solvent Isotope Effect on the Stability of a Heterodimeric Protein

Abstract Protein stability arises from a fine balance between stabilizing forces such as hydrophobic interactions, hydrogen bonding, and ionic interactions, and destabilizing contributions from solvent exposure and electrostatics. Although hydrophobic burial is the dominant driving force for folding, intra-chain hydrogen bonds and ionic interactions modulate stability in context-dependent ways, with effects that vary depending on their location and environment within the protein. Most studies of protein stability have focused on perturbations induced by pH, solvent composition, or mutations in protonated water, leaving the influence of solvent isotopes relatively underexplored. Notably, despite stronger hydrogen bonding in D 2 O, proteins exhibit diverse stability responses upon transfer from H 2 O to D 2 O, suggesting that differential hydration of nonpolar groups plays a key role. Here, the solvent isotope effect on protein stability is investigated using double-chain monellin (dcMN), a β-sheet–rich, two-chain protein with well-characterized folding behavior. By combining conventional equilibrium unfolding measurements with hydrogen-deuterium exchange mass spectrometry (HDX-MS), the stability of wild-type and a less hydrophobic mutant (C42A) dcMN was compared in H 2 O and D 2 O, revealing greater stabilization of the wild-type protein in D 2 O and highlighting the importance of hydrophobic interactions in governing isotope-dependent stability.