Distinct Solvent Effect on the Conformation and Charge State Distribution of Typical Proteases Revealed by Ion...

Solvent effects on proteins serve as intrinsic determinants of their folding conformation, dynamic behavior, and molecular recognition. This study systematically analyzed the effects of four polar solvents, including dimethylformamide (DMF), dimethylacetamide (DMA), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO, with elevated polarities), on three prototype proteases by ion mobility mass spectrometry (IM-MS). The results demonstrated that changes in solvent type and concentration could significantly alter the gas-phase conformation of proteases, inducing varying degrees of partial unfolding and the formation of intermediate states. DMF, NMP, and DMA exhibited no significant denaturing effect on protein conformations. In contrast, DMSO with the highest polarity tended to form folded states at lower concentrations and unfolded states at higher concentrations. Further analysis revealed that the trend of collision cross-section (CCS) changes did not fully align with the patterns of the charge state, suggesting that solvent environments may regulate protein conformational diversity and charge distribution through distinct mechanisms. DMSO has been demonstrated to promote the adoption of unfolded conformations by proteins that go beyond their native state. In contrast, the other three solvents have been shown to drive the protein conformation closer to its native state without inducing excessive denaturation. This study not only reveals the influence of polar solvents on the stability and kinetics of proteases but also provides a new experimental basis for understanding the protein-solvent interaction.