Unusual Hydrations of Amide I: An Insight into Protein Structure and Flexibility

The higher atomic mass of deuterium basically affects hydrogen-bonding interactions and solvent association, raising critical alarms about the precision of biomolecular measurements executed in heavy water. Herein, we combine linear infrared (IR) spectroscopy, circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations to explore how solvent isotopic exchange alters protein structure as well as dynamics. Comparative studies of different protonated (H 2 O, CH 3 OH) and deuterated (D 2 O, CD 3 OD) solvents expose noticeable differences in hydrogen-bond lifetimes, solvation patterns, and protein secondary structural constancy. Particularly, the amide I hydrogen-bonded complex shows suggestively longer lifetimes in D 2 O than in H 2 O, reflecting slower hydrogen-bond dynamics and reduced flexibility of the protein backbone. Similar effects are detected in methanol/methanol-d 4, also highlighting that these phenomena are not unique to water but are intrinsic to deuterium replacement. These multitechnique results clearly validate that biomolecular structures and dynamical behaviors in deuterated solvents are markedly different from those in their protonated surroundings. Our conclusions extend the understanding of isotope substitution effects in solvation and underscore the necessity for careful interpretation of experimental data acquired in D 2 O or other deuterated solvents, mainly when concluding native biological conditions.