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.

The molecular conformation of the carboxyl group can be crucial for its chemical properties and intermolecular interactions, especially in complex molecular environments such as polypeptides. Here, we study the conformational behaviour of the model amino acid N-acetylproline in solution at room temperature with two-dimensional infrared spectroscopy. We find that the carboxyl group of N-acetylproline adopts two distinct conformations, syn- and anti-. In the syn-conformer the O–H group is oriented at ~60^∘ with respect to the C=O and in the anti-conformer the O–H is anti-parallel to the C=O. In hydrogen-bond accepting solvents such as dimethyl sulfoxide or water, we observe that, similar to simple carboxylic acids, around 20% of the -COOH groups adopt an anti-conformation. However, when N-acetylproline is dissolved in a weakly hydrogen-bond accepting solvent (acetonitrile), we observe the formation of a strong intramolecular hydrogen bond between the carboxyl group in the anti-conformation and the amide group, which stabilizes the anti-conformer, increasing its relative abundance to ~60%.

We used two-dimensional infrared spectroscopy to disentangle the broad infrared band in the amide II vibrational regions of Bombyx mori native silk films, identifying the single amide II modes and correlating them to specific secondary structure. Amide I and amide II modes have a strong vibrational coupling, which manifests as cross-peaks in 2D infrared spectra with frequencies determined by both the amide I and amide II frequencies of the same secondary structure. By cross referencing with well-known amide I assignments, we determined that the amide II (N-H) absorbs at around 1552 and at 1530 cm^–1 for helical and β-sheet structures, respectively. We also observed a peak at 1517 cm⁻¹ that could not be easily assigned to an amide II mode, and instead we tentatively assigned it to a Tyrosine sidechain. These results stand in contrast with previous findings from linear infrared spectroscopy, highlighting the ability of multidimensional spectroscopy for untangling convoluted spectra, and suggesting the need for caution when assigning silk amide II spectra.

The macroscopic mechanical properties of biological hydrogels are broadly studied and successfully mimicked in synthetic materials, but little is known about the molecular interactions that mediate these properties. Here we use two-dimensional infrared spectroscopy (2D-IR) to study the pH-induced gelation of hyaluronic acid, a ubiquitous biopolymer, which undergoes a transition from a viscous to an elastic state in a narrow pH range around 2.5. We find that the gelation originates from the enhanced formation of strong inter-chain connections, consisting of a double amide-COOH hydrogen-bond and an N-D-COO- hydrogen-bond on the adjacent sugars of the hyaluronan disaccharide unit. We confirm the enhanced inter-chain connectivity in the elastic state by AFM imaging.

The remarkable elastic properties of polymers are ultimately due to their molecular structure, but the relation between the macroscopic and molecular properties is often difficult to establish, in particular for (bio)polymers that contain hydrogen bonds, which can easily rearrange upon mechanical deformation. Here we show that two-dimensional infrared spectroscopy on polymer films in a miniature stress tester sheds new light on how the hydrogen-bond structure of a polymer is related to its viscoelastic response. We study thermoplastic polyurethane, a block copolymer consisting of hard segments of hydrogen-bonded urethane groups embedded in a soft matrix of polyether chains. The conventional infrared spectrum shows that, upon deformation, the number of hydrogen bonds increases, a process that is largely reversible. However, the 2DIR spectrum reveals that the distribution of hydrogen-bond strengths becomes slightly narrower after a deformation cycle, due to the disruption of weak hydrogen bonds, an effect that could explain the strain-cycle induced softening (Mullins effect) of polyurethane. These results show how rheo-2DIR spectroscopy can bridge the gap between the molecular structure and the macroscopic elastic properties of (bio)polymers.

Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water–collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H 2 O/D 2 O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H 2 O and D 2 O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D 2 O than in H 2 O, and collagen in D 2 O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H 2 O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D 2 O is less hydrated than in H 2 O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen–water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly.