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

Abstract

D 2 O is commonly used as a solvent instead of H 2 O in spectroscopic studies of proteins, in particular, in infrared and nuclear-magnetic-resonance spectroscopy. D 2 O is chemically equivalent to H 2 O, and the differences, particularly in hydrogen-bond strength, are often ignored. However, replacing solvent water with D 2 O can affect not only the kinetics but also the structure and stability of biomolecules. Recent experiments have shown that even the mesoscopic structures and the elastic properties of biomolecular assemblies, such as amyloids and protein networks, can be very different in D 2 O and H 2 O. We discuss these findings, which probably are just the tip of the iceberg, and which seem to call for obtaining a better understanding of the H 2 O/D 2 O-isotope effect on water−water and water−protein interactions. Such improved understanding may change the differences between H 2 O and D 2 O as biomolecular solvents from an elephant in the room to an opportunity for protein research.

Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration

“…These findings are consistent with previous studies which have shown that D 2 O is a poorer protein solvent than H 2 O, and that D 2 O favors a less water-exposed but more stable protein structure ( 38 , 57 – 66 ), affecting the assembly properties ( 19 , 20 , 67 ) (see ref. 68 for more details).…”

“…Turbidity data; IR and 2D-IR data; TEM images; cryo-TEM images; CRM images; coarse grained simulations; molecular dynamics simulation data have been deposited in UvAauas.figshare ( https://doi.org/10.21942/uva.24829896 ) ( 102 ).…”

Using 2D-IR Spectroscopy to Measure the Structure, Dynamics, and Intermolecular Interactions of Proteins in H2O

“…Changes to the amide I band of a protein upon drug binding contain information relating to how the secondary structure and local dynamics are modified by the interaction. Applications of 2D-IR amide I spectroscopy in D 2 O have demonstrated significant drug-induced changes, which correlate with function of the drug molecule. , These effects were often associated with alterations in the dynamics of the protein rather than large-scale secondary structure changes, and thus, their detection relies on the sensitivity of 2D-IR to factors such as transition dipole moments or anharmonicities, which evade IR absorption measurements. , Differences in the H-bonding ability of H 2 O and D 2 O mean that H/D exchange may affect the balance of competitive solvation of the drug molecule and protein binding site, so measurements in the native solvent will be important while also removing the practical need to exchange buffer solutions or express proteins in deuterated media.…”

“… 19 , 48 These effects were often associated with alterations in the dynamics of the protein rather than large-scale secondary structure changes, and thus, their detection relies on the sensitivity of 2D-IR to factors such as transition dipole moments or anharmonicities, which evade IR absorption measurements. 19 , 48 Differences in the H-bonding ability of H 2 O and D 2 O mean that H/D exchange may affect the balance of competitive solvation of the drug molecule and protein binding site, so measurements in the native solvent will be important 49 while also removing the practical need to exchange buffer solutions or express proteins in deuterated media.…”

Effects of Aqueous Isotopic Substitution on the Adsorption Dynamics and Dilational Rheology of β-Lactoglobulin Layers at the Water/Air Interface

“…The use of heavy water (D 2 O) as an aqueous medium is a prerequisite for advanced protocols in various experimental characterization methods, for example, neutron scattering and some spectroscopic techniques such as nuclear magnetic resonance and infrared spectroscopy. − The use of D 2 O instead of H 2 O in aqueous protein bulk studies was very recently discussed in a perspective article by Giubertoni et al The basis of the differences between the solvation effects of H 2 O and D 2 O is that the hydrogen bond (OH ··· O) is slightly weaker than the “hydrogen bond” in D 2 O (OD ··· O). Such a solvation effect may influence to a certain degree the stability/flexibility of protein globules, − which, in turn, may lead to changes in some physicochemical properties of globular proteins either in the bulk or when adsorbed at interfaces, for instance, the denaturation and aggregation kinetics in the bulk − as well as the adsorption kinetics at interfaces. , The observed inhibitory effect of D 2 O on the activity of some enzymes is worth noting …”