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

D2O is commonly used as a solvent instead of H2O in spectroscopic studies of proteins, in particular, in infrared and nuclear-magnetic-resonance spectroscopy. D2O is chemically equivalent to H2O, and the differences, particularly in hydrogen-bond strength, are often ignored. However, replacing solvent water with D2O 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 D2O and H2O. 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 H2O/D2O-isotope effect on water–water and water–protein interactions. Such improved understanding may change the differences between H2O and D2O as biomolecular solvents from an elephant in the room to an opportunity for protein research.


■ INTRODUCTION
D 2 O, or heavy water, is a stable isotopomer of H 2 O, containing deuterium instead of the most common hydrogen isotope protium.Deuterium was discovered in 1931 by H. Urey, 1 who was awarded the Nobel Prize for this finding in 1934.The chemical and physical properties of D 2 O were first studied by G. Lewis and co-workers in the early 1930s 2,3 and are very similar to those of H 2 O (Table 1).For this reason, D 2 O is often used as a solvent instead of H 2 O in experiments where the H atoms of water form a problem, such as in nuclear magnetic resonance, neutron scattering, and infrared spectroscopy and imaging.This holds in particular for studies of biomolecules: in both protein NMR and infrared spectroscopy and imaging, 4−6 it is standard practice to use D 2 O as a solvent.In the case of infrared spectroscopy, this is done because the vibrational modes of the amide groups, which carry crucial information on the protein structure, 7 have spectral overlap with the bending mode of H 2 O (both are in the 1600−1700 cm −1 frequency range).The D 2 O-bending frequency is 1250 cm −1 , eliminating the overlap problem and making D 2 O the seemingly perfect replacement of H 2 O.
The effect of H/D substitution on the kinetics of chemical reactions is well-known, and has been extensively studied and applied, for instance to study reaction mechanisms 12 and to monitor protein folding. 13Interestingly, recent work shows that the kinetic effects induced by substituting D 2 O for H 2 O might also be useful for biomedical purposes: 14 epithelial cells grown in a medium containing 45% D 2 O show significantly reduced migration and proliferation rates (Figure 1), and a similar slowdown in dynamics was observed in other cells, 15−17 an effect that might find use for the storage of biological materials such as organs, or for anticancer treatment. 15hile the effect of H/D substitution on kinetics is well established, it is often (implicitly) assumed that the effect of H 2 O/D 2 O substitution on the structure of biomolecules and biomolecular assemblies is small.−41 The fact that H 2 O/D 2 O replacement can change the structure of biopolymers and their assemblies should not come as a complete surprise: the hydrogen-bond a structures of H 2 O and D 2 O are known to be different, 42 and hydration and the hydrophobic effect are essential for all biomacromolecules, ranging from polysaccharides to proteins.Hydrating water molecules create a water network around solutes that not only acts as structure stabilizer but also mediates intra-and intermolecular interactions.As stated recently by Fischer et al., 43 hydration represents "an additional evolutionary constraint upon protein sequence to maintain ligand binding and modulate the affinity of those interactions", to which we might add that since evolution has optimized protein structure and dynamics in H 2 O rather than D 2 O, and since the hydrogen-bond structures of these two liquids are different, differences in structure and dynamics are to be expected when replacing one with the other.
The H 2 O/D 2 O-induced changes in biomolecular structure seem to call for more detailed studies of the difference between liquid D 2 O and H 2 O, but they also suggest fascinating new research opportunities.In this Perspective, we first briefly describe the differences between H 2 O and D 2 O; then we summarize and discuss the existing experimental evidence for isotope-induced structural changes in biomolecules and biomolecular assemblies; finally, we discuss the current challenges and perspectives, in particular the possibility of using D 2 O to investigate the role of hydration in protein stability and interactions.
The interplay of nuclear quantum effects (NQEs) underlying the physical and chemical differences between liquid D 2 O and H 2 O is quite subtle.Simply put, the low mass of the hydrogen atom makes it behave more as a delocalized quantum particle than the heavier deuterium.This delocalization can have a substantial effect on the hydrogen bond strength. 10 The larger distance spread for H vs D leads to a strengthening of the H-bond, while the larger angular spread leads to a weakening.Hence, these two nuclear quantum effects have contrary consequences for the H-bond strength.Depending on the details of the H-bond, one or the other effect may dominate, resulting in a weakening or strengthening of Hbonds upon isotopic substitution.Short hydrogen bonds are typically strengthened due to NQEs, whereas long ones are weakened. 10Here we summarize the most important differences that are generally agreed upon in the literature, focusing on the points that are relevant for understanding how replacing H 2 O with D 2 O can change the structures of biomolecules and biomolecular assemblies.
The structure of liquid D 2 O and water has been investigated using different methods, in particular X-ray, γ-ray, and neutron scattering.By combining X-ray measurements with molecular simulations, it was found that the covalent bond between oxygen and protium (O−H) is 3% longer with respect to the one between oxygen and deuterium (O−D), see Figure 2 (neutron scattering studies indicate a somewhat smaller isotope effect on the covalent bond length 10 ).In D 2 O, the hydrogen-bond network is more tetrahedral than that in H 2 O and the hydrogen-bond coordination number is higher, 42 both effects indicating stronger hydrogen bonds and a more structured hydrogen-bond network.The average hydrogenbond distance (the O•••O distance of two hydrogen-bonded water molecules) is 4% longer in D 2 O, as is also reflected in its lower molar density compared to H 2 O (cf. the situation in ice, where the hydrogen bonds are also stronger than in liquid water).In ab initio calculations on hydrogen-bonded oligomers, it was also found that the hydrogen-bond strength is 0.2−0.3kcal/mol larger in D 2 O than in H 2 O. 44 Finally, the    18−21 For instance, using differential scanning calorimetry (DSC), it was found that the denaturation temperature of lysozyme and BSA is 2−3 °C higher in heavy water than in water. 18Circular dichroism (CD) experiments, which are more structure-sensitive than DSC measurements, showed that the onset temperature of the irreversible thermal denaturation (i.e., the temperature of the irreversible change of the secondary structure) of BSA is 58 19 Upon heat-treatment at 65 °C, BSA also retains a larger percentage of monomers in heavy water than in water (85% versus 75%, respectively), again indicating that the BSA monomeric form is more stable in D 2 O. 20 Similar results have been found for other, nonglobular proteins, such as acyl carrier proteins, 22 collagen, 24 ribonuclease A 27 and Drosophila signal-transduction protein Drk. 26imilar enhanced stability of the folded state was also observed for κ-carrageenan, which undergoes to a liquid-to-gel transition by forming double helices, that are stabilized significantly more in D 2 O. 25 The increased stability of folded and native structures in D 2 O indicates a stronger tendency to adopt a more compact, less solvent-exposed conformation in this solvent.For instance, a D 2 O-induced tightening of the helical structure has been proposed for actin, based on combined rheological and fluorescence experiments. 14 ≈ 3 °C elastin-like peptides 28 DSC, CD, IR enhanced stability of the collapsed state, Propensity to form β-turn/β-aggregate, LCST H2O − LCST D2O ≈ 2−5 °C peptides containing alanine 29 CD propensity for PPII structure: 5−200% higher PPII signal in D 2 O plastocyanin 32 MD altered solvent−protein interactions: 10−30% reduction of protein−water H-bonds test polypeptides 34 MD altered solvent−protein interactions agarose (Ag2) 33 NMR lower solvent−polysaccharide affinity, N w H2O /N w D2O ≈ 3.8 ribonuclease T1 31 luminescence increased protein rigidity, IPL D2O = 36 ms, IPL H2O = 28 ms β-lactoglobulin 31 luminescence increased protein rigidity, IPL D2O = 44 ms, IPL H2O = 30 ms liver alcohol dehydrogenase 31 luminescence increased protein rigidity IPL D2O = 819 ms, IPL H2O = 630 ms alkaline phosphatase 31 luminescence increased protein rigidity, IPL D2O = 2142 ms, IPL H2O = 2060 ms apo-azurin 31 luminescence increased protein rigidity IPL D2O = 603 ms, IPL H2O = 564 ms TAS1R2/TAS1R3 receptor 30 MD smaller radius of gyration R g D2O is ≈3% smaller than R g H2O azurin, 35 29 Since it is believed that the PP II conformation perturbs the bulk hydrogen-bond network of the surrounding water less strongly than does an α-helical conformation, this effect was attributed to the increased energetic cost of protein solvation in D 2 O.The difference in protein stability and the preference for PP II structure suggest that interactions between solvent and protein might be modified in D 2 O compared to H 2 O, leading to changes in the intraprotein hydrogen-bond network.This possibility has been investigated mostly by means of molecular dynamics simulations of various biomolecules, such as plastocyanin, 32 RNA hairpins, 23 and peptides. 34Interestingly, in ref 32, it was observed that a reduction of the number of hydrogen bonds between solvent and protein occurs mostly when polar and positively charged side groups are involved, while the opposite is observed for negatively charged side groups.Overall, however, a 10−30% reduction in the number of water molecules engaged in hydrogen bonds with the protein was observed in D 2 O compared to H 2 O, which was correlated to the enhancement of intramolecular interactions in this solvent. 32A lower affinity between D 2 O and solute was also observed in NMR studies on agarose. 33The increased rigidity which Cioni et al. have observed for different proteins (see Table 2) also supports the idea that protein−solvent interactions are altered in D 2 O: 31 using luminescence methods it was found for 5 proteins out of the 7 analyzed that D 2 O increases protein rigidity, with a protein-dependent rigidity enhancement.In this respect it is interesting to note that some proteins crystallize more efficiently in D 2 O than in H 2 O, 46 a phenomenon that in the case of ref 46 was even accompanied The Journal of Physical Chemistry B by a difference in crystal symmetry and structure (whereas in general protein crystal structures seem to be independent of whether H 2 O or D 2 O is used 47−49 ).The D 2 O-induced damping of conformational fluctuations can be attributed to stronger solvent−solvent interactions, 31 which reduce protein hydration and promote intramolecular interactions (as was observed in ref 32).The reduction in structural fluctuations in D 2 O may thus be explained by the fact that water−protein interactions can destabilize proteins by lowering the freeenergy barriers between different conformations.
We conclude our list of proteins with the well-known and intriguing fact that D 2 O tastes sweet.A recent moleculardynamics study of this isotope effect by the Jungwirth group 30 has shown that the transmembrane part of the human sweettaste sensor protein is more compact, stiffer, and subject to less structural fluctuations in D 2 O than in H 2 O (Figure 3).This study again supports the idea of a reduction in protein hydration in D 2 O compared to H 2 O. Indeed, in a more recent study the same group has found that in D 2 O, water has a stronger propensity to form water/water hydrogen bonds than water/amino-acid hydrogen bonds (interestingly, this behavior does not follow the hydrophobicity scale of the amino acids). 35t was also found that globular proteins (azurin, lactoglobulin, and ribonuclease) are significantly more compact in D 2 O than in H 2 O. Jungwirth et al. conclude that "D 2 O is a somewhat worse solvent for biomolecules than H 2 O.This also implies that association between proteins or between a protein and a biomembrane may be positively affected by water deuteration".In the next section, we will see experimental results that support this idea.D 2 O-Induced Changes in Protein Assemblies and Networks.We have seen that D 2 O increases the stability of the folded state of proteins, in particular promoting the formation of secondary structures that least disrupt the hydrogen-bond network of water, and that protein hydration is reduced in D 2 O.More recently, it has become clear that these changes at the molecular level can affect the propensity and mechanisms of aggregation/assembly of biopolymers into larger supramolecular structures, leading to different mechan-ical and thermodynamic properties of the final aggregate/ assembly (Table 3).In particular, Salvatella et al. have found that androgen receptors have a stronger tendency to form biomolecular condensates by liquid−liquid phase separation (LLPS) in D 2 O than in H 2 O. 36 Interestingly, in this study, it was shown that replacing less than 10% water (as is common in NMR) with D 2 O can already significantly affect the phase equilibrium of the condensation, with a decrease of the cloud point by 0.5 °C for each added percent of D 2 O, and that the size of the condensates becomes larger with increasing amount of added D 2 O.These changes were attributed to the enhancement in D 2 O of the intermolecular interactions that drive the initial oligomerization.Similarly, an elegant study by Beckett et al. has shown that the dimerization of the Escherichia coli protein BirA is more favorable in D 2 O than in H 2 O, with a dimer dissociation constant that is 10 times smaller in the former. 50A similar D 2 O-induced alteration of the aggregation propensity (and possibly the final aggregate size) has been proposed for BSA aggregates, based on thioflavin fluorescence, turbidity, and circular dichroism experiments. 19,20,51everal studies have shown a significant difference in protein assembly rates in water and D 2 O, with assembly occurring faster in the latter.For instance, the aggregation and simultaneous double-helix formation of κ-carrageenan occurs faster in D 2 O than in H 2 O. 25 Faster aggregation in D 2 O was also observed for gelatin, 37 casein, 38 and bovine serum albumin. 19These examples all show faster assembly in D 2 O, but self-assembly processes can also become slower in D 2 O. Recently, a ground-breaking study by Cho et al. has shown that amyloid formation of insulin occurs slower in D 2 O than in H 2 O (Figure 4). 39This effect was attributed to the presence of intermediates that adopt intermolecular beta-sheet structures, which are more favored in D 2 O than in H 2 O. Using D 2 O as a solvent instead of H 2 O increases the free-energy barrier for unfolding these intermediates, which is a necessary step for the final fibril formation.A similar enhancement of oligomer stability in heavy water was suggested for transthyretin tetramer. 49Interestingly, it was recently found that the fibrillization of alpha-synuclein (the protein responsible for Abbreviations: K dim = equilibrium dissociation constant for dimerization; τ lag = lag time; G′ = elastic modulus at a frequency of 1 Hz; r = rate of initial gelation; G = shear modulus; DCR = derived count rate (light-scattering intensity); Gel.On.= gelation onset; τ = initial turbidity; SE = sedimentation equilibrium measurements; 2DIR = two-dimensional infrared spectroscopy; SAXS= small angle X-ray scattering; SANS = smallangle neutron scattering.b Two methods were used to induce gelation, referred to as RG and TG.

The Journal of Physical Chemistry B pubs.acs.org/JPCB Perspective
Parkinson's disease) proceeds faster in D 2 O than in water. 40his acceleration was attributed to enhanced protein−protein interactions in D 2 O that facilitate the refolding of alphasynuclein, which is required for initiating its fibrillization.Surprisingly, not only protein-assembly kinetics but even the viscoelastic properties of biopolymer networks can be different in D 2 O and H 2 O.The mechanical properties of reconstituted actin networks are affected by using D 2 O instead of H 2 O: in D 2 O the actin filaments behave as a transiently cross-linked network rather than the typical behavior of an entangled network (as is observed in H 2 O).This peculiar behavior in D 2 O was recently explained by the finding that D 2 O induces the formation of multifilament bundles, leading to a structural reorganization of the actin network and different mechanical properties. 52The difference in the network structure was attributed to a larger stickiness between actin filaments in D 2 O because of enhanced intermolecular interactions in this solvent. 14,52Similarly, the elastic modulus of gels formed by the aggregation of κ-carrageenan is ∼10−20% higher in D 2 O than in H 2 O because of the larger number of cross-links formed between the chains. 25Such increased network rigidity has also been observed in gelatin and casein gels. 37,38In contrast, Brenner et al. found that in agarose gel the mechanical properties are the same in D 2 O and H 2 O, even though D 2 O does enhance the stability of the helical structure and gives rise to gels with larger heterogeneity on the micrometer scale (and not the nanometer scale). 33Finally, an intriguing topological difference in biopolymer-network structure has been found in the case of pectin, for which recent experiments have shown that the fractal dimension of the gel network formed is higher in D 2 O than in H 2 O (indicating that in D 2 O the gel is more clustered), 41 an observation that again "highlights the need to be mindful of changes induced when substituting D 2 O in systems with significant hydrogen bonding". 41he Origin of D 2 O-Induced Changes in Stability and Structure.In D 2 O, biopolymers are exposed to a more strongly hydrogen-bonded water network, 42 and therefore creating a solvation cavity to accommodate the protein (or increasing the solvent-exposed surface area of a protein) is energetically less favorable in D 2 O because of the additional enthalpic cost required to break the water hydrogen bonds.This energetic loss is enhanced when the solvent needs to reorganize around nonpolar groups, and hence hydrophobic patches have a stronger tendency to cluster in D 2 O than in H 2 O, an effect we may refer to as isotopically enhanced hydrophobic effect.However, a theoretical analysis by Graziano and Pica has shown that the H 2 O/D 2 O effect on the hydrogen-bond structure may not be sufficient to explain D 2 O-enhanced protein stability. 11Due to the lower molecular polarizability of D 2 O, van der Waals attractive interactions are less favorable in D 2 O, and thus fewer interactions take place between protein and water.Reduced van der Waals interactions affect the binding affinity of D 2 O to biomacromolecules, which may lead to changes in the hydration shell surrounding the biomolecules. 23,35The combination of reduced van der Waals interaction and the higher enthalpic cost of water−water hydrogen-bond breaking will likely change the hydration capability of D 2 O with respect to H 2 O in a synergistic way.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. 47,49A decrease in water−protein interaction in D 2 O compared to H 2 O is also consistent with the enhanced rigidity observed, for instance, in collagen peptides, where intramolecular hydrophobic interactions are minimal and thus enhanced hydrophobic effect alone cannot explain the increased rigidity. 24e have seen that biomolecular assembly can occur at different rates in D 2 O and H 2 O (Table 3 and Figure 4).If the aggregation is driven by hydrophobic or hydrophilic interactions, the kinetics are expected to be different in D 2 O.As discussed before, D 2 O enhances the hydrophobic interactions (enhancing the aggregation) and has reduced protein hydration compared to H 2 O.This latter effect implies that the desolvation enthalpy, i.e., the energy required to break the hydrogen bonds between water and hydrophilic groups to allow the formation of bonds between hydrophilic groups, is lower in D 2 O than in H 2 O.This is consistent with the faster assembly rate reported for several systems. 25,37,38However, if the aggregation process involves the formation of intermediates stabilized by hydrophobic interactions, the assembly might be slower in D 2 O, as observed in the case of amyloid formation. 39o form fibrils, intermediates have to undergo partial unfolding, a process that is energetically more unfavorable in D 2 O since the intermediates are more stable due to the enhanced hydrophobic effect.

■ FROM ELEPHANT IN THE ROOM TO OPPORTUNITY FOR PROTEIN RESEARCH
Although in general replacing H 2 O with D 2 O has a limited effect on protein structure (as is demonstrated by the large number of successful studies in which this procedure was used), the experiments and simulations discussed above show that replacing H 2 O with D 2 O can in some cases significantly change the structure and stability of proteins and protein assemblies.On the one hand, this means that experiments on  Thus, we believe that the difference in biopolymer hydration in H 2 O and D 2 O can be exploited to gain a better understanding of biopolymers, in particular, of biopolymer− solvent interactions and their role in defining the structure and dynamics of proteins and protein assemblies.This constitutes an interesting next challenge for the scientific community working on proteins and protein assemblies.■ ADDITIONAL NOTE a We will use the term "hydrogen bond" to denote both hydrogen bonds and deuterium bonds.
Specifically, for an O−H•••O hydrogen bond, the hydrogen-bond strength is a function of the O•••O distance (the shorter, the stronger) and the O−H•••O bond angle (the straighter, the stronger).

Figure 1 .
Figure 1."Cells in slow motion." 14(a) Epithelial cell proliferation is much slower in a D 2 O-rich medium.The duration of the cell cycle is roughly 17 h under normal (H 2 O) conditions and 44 h in water containing 45% D 2 O.(b) Independent of the culturing conditions, the actin cytoskeleton (orange) and the nucleus (blue) remain intact.Shown are typical pictures, which suggest that structures are comparable for the different H 2 O and D 2 O concentrations.Figure adapted from ref 14, Copyright 2021 Schnauß et al. (licensed under CC BY-NC 4.0).

Figure 2 .
Figure 2. Average lengths of the covalent and hydrogen bonds in liquid H 2 O (a) and D 2 O (b).
a Abbreviations: T d = denaturation temperature; Irr.T d = irreversible denaturation temperature; T m = melting temperature of the native state; T 0 = transition temperature from folded-to-unfolded; R g = radius of gyration; IPL = intrinsic Trp phosphorescence lifetime; ΔG N→U = Gibbs energy of unfolding; T gel→liq = gel-to-liquid transition temperature; LCST = lower critical solution temperature; N w = number of hydration waters per mass unit of agarose; DSC = differential scanning calorimetry; SE-HPLC = size exclusion high-performance liquid chromatography; CD = circular dichroism; DLS = dynamic light scattering; Fl = fluorescence measurements; NMR = nuclear magnetic resonance; MD = molecular dynamics simulations.

Figure 3 .
Figure 3. Differences between the behavior of the transmembrane part of the human sweet taste receptor in H 2 O vs D 2 O. (a) Structure of the TMD of the TAS1R2/TAS1R3 receptor with the probability density (volumetric map) of H 2 O (blue) or D 2 O (red) molecules within 10 Å of the protein.The conserved water molecules in the X-ray templates are shown in cyan.Water molecules predicted with the software OpenEye52 are shown in licorice representation.(b) Time evolution of the radii of gyration in H 2 O (blue) and D 2 O (red) from three microsecond time scale simulations (separated by vertical dashed lines) with total mean values as dashed lines, showing that the protein is more compact in D 2 O. (c) Snapshot of the transmembrane part of the human sweet taste receptor color-coded that red/blue represents parts more/less rigid in D 2 O vs H 2 O.The embedding lipid membrane is represented in gray.(d) Difference in root-mean-square fluctuations in MD trajectories.Negative/positive values mean that structures are more/less rigid in D 2 O than in H 2 O.The red line represents the sum of all residues.INT, intracellular; EXT, extracellular.Adapted from ref 30, copyright 2021 Ben Abu et al. (licensed under CC BY).

Figure 4 .
Figure 4. Insulin (INS) fibrillization kinetics in H 2 O and D 2 O and proposed fibrillization mechanism explaining the slower assembly in D 2 O. Reproduced from ref 39, Chun et al., published by the Royal Society of Chemistry (licensed under CC BY-NC 3.0).

■
AUTHOR INFORMATION Mischa Bonn is a Director at the Max Planck Institute for Polymer Research since 2011, heading the Molecular Spectroscopy Department.Mischa completed his M.Sc.degree in physical chemistry in 1993 at the University of Amsterdam and performed his Ph.D. research at the FOM-Institute for Atomic and Molecular Physics in Amsterdam, where he shared an office with (and had to listen to the music of) Sander Woutersen.After postdoctoral stays at the Fritz Haber Institute and Columbia University, he went to Leiden University and returned to the Institute for Atomic and Molecular Physics as group leader in 2004.His scientific interests focus on the development and application of ultrafast spectroscopies to study natural phenomena, specifically at interfaces and often involving Mischa's favorite molecule: water.Sander Woutersen obtained his M.Sc. in physical chemistry at the University of Amsterdam (1995) and did his Ph.D. research at the FOM-Institute for Atomic and Molecular Physics in Amsterdam, where he shared an office with (and was regularly made fun of by) Mischa Bonn.After a postdoctoral fellowship with Peter Hamm at the Max Born Institute in Berlin, he returned to Amsterdam, where he eventually became professor in physical chemistry.Sander's research is at the interface between spectroscopy and soft matter.■ ACKNOWLEDGMENTS We thank Ioana Ilie for fruitful discussions.This publication is part of the project (with Project Number VI.Veni.212.240) of the NWO Talent Programme Veni 2021, which is financed by the Dutch Research Council (NWO).

Table 1 .
Selected Physical and Chemical Properties of H 2 O and D 2 O 8−11

Table 2 .
Effects of D 2 O on the Properties of Proteins and Other Biomolecules a Similarly, Cremer et al. have shown that elastin-like polypeptides (ELPs) undergo a hydrophobic collapse that is accompanied by the formation

The Journal of Physical Chemistry B pubs.acs.org/JPCB Perspective proteins
58,59ich H 2 O has been replaced with D 2 O should be interpreted with caution.On the other hand, the possibility of "tuning" the hydration strength by varying the isotopic composition provides a unique tool to investigate protein hydration, and might be useful for gaining a better understanding of the role of water in defining protein structure.Water strongly influences the properties of proteins and is also believed to regulate and mediate protein−protein/ligand interactions in many biopolymers, such as collagen or silk fibroin, and water is also believed to play a crucial role in determining collagen interactions with minerals in bone tissue.54Experimentsdesigned to investigate protein hydration usually measure how the protein properties change upon varying the solvent, for instance, by replacing or mixing water with an organic solvent.This clearly changes the protein hydration but unfortunately also modifies many other solvent properties, such as the dielectric constant and the molecular size, which might affect protein intra-and intermolecular interactions.Replacing water with D 2 O is a unique method to specifically modify the water hydrogen bonding without changing the other solvent properties.Comparing protein behavior in H 2 O and D 2 O and their mixtures thus constitutes an elegant way to determine specifically the contribution of water hydrogen bonding to the physical and chemical properties of proteins without having to resort to changes in the solvent that alter more than the protein hydration.Such D 2 O vs H 2 O experiments may not always be easy to realize, but for instance two-dimensional infrared spectroscopy on proteins in H 2 O has already been reported.39,55−57Thisrecentadvancementenablesresearchersto study proteins in more natural systems, such as in cells or in blood serum.58,59SinceOenhances the hydrophobic effect, a comparison of protein secondary structure in H 2 O and D 2 O can reveal the role of hydrophobic interactions in the stabilization of the proteins or in promoting their collapse.Similarly, comparing self-assembly kinetics in water and D 2 O can be a valuable method to gain a better understanding of the aggregation process, in particular in the case of fibril formation.Fibril formation can occur spontaneously via a nucleation-andgrowth mechanism(1-step-nucleation or 1SN) or in two steps via the formation of intermediate aggregates (2SN) stabilized by hydrophobic effects.Intermediates subsequently need to undergo structural transformations to attain the fibrillar conformation, representing the rate-limiting step for fibrillization.Since D 2 O stabilizes hydrophobic interactions, the aggregation rate in D 2 O with respect to H 2 O is reduced if the mechanism involves intermediates, because their unfolding is energetically more unfavorable in D 2 O. Comparing the fibrillization rate in water and D 2 O can therefore reveal whether intermediates are present and hence if the amyloid formation occurs by a 2SN or 1SN mechanism.On the same note, the ability of D 2 O to slow the aggregation and stabilize the intermediates can be used to study the intermediate species.Intermediates are transient and metastable aggregates, which are quite challenging to detect and characterize structurally.By using D 2 O, we can follow the protein selfassembly in "slow motion".