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

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[Formula: see text]O/D[Formula: see text]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[Formula: see text]O and D[Formula: see text]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[Formula: see text]O than in H[Formula: see text]O, and collagen in D[Formula: see text]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[Formula: see text]O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D[Formula: see text]O is less hydrated than in H[Formula: see text]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.

Nanoconfined Water Clusters in Zinc White Oil Paint.

Pigments in oil paint are bound by a complex oil polymer network that is prone to water-related chemical degradation. We use cryo-Fourier-transform infrared spectroscopy and differential scanning calorimetry to study how water distributes inside zinc white oil paint. By measuring water freezing and melting transitions, we show that water-saturated zinc white oil paint contains both liquid-like clustered water and nonclustered water. A comparison of titanium white paint and nonpigmented model systems indicates that water clustering happens near the pigment-polymer interface. The cluster size was estimated in the nanometer range based on the ice melting and freezing temperatures and on the position of the O-D vibration band. As liquid-like water can play a crucial role in the dissolution and transport of ions and molecules, understanding the factors that favor this phenomenon is essential for establishing safe conditions for the conservation of painted works of art.

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.

Raman Diffusion-Ordered Spectroscopy.

The Stokes-Einstein relation, which relates the diffusion coefficient of a molecule to its hydrodynamic radius, is commonly used to determine molecular sizes in chemical analysis methods. Here, we combine the size sensitivity of such diffusion-based methods with the structure sensitivity of Raman spectroscopy by performing Raman diffusion-ordered spectroscopy (Raman-DOSY). The core of the Raman-DOSY setup is a flow cell with a Y-shaped channel containing two inlets: one for the sample solution and one for the pure solvent. The two liquids are injected at the same flow rate, giving rise to two parallel laminar flows in the channel. After the flow stops, the solute molecules diffuse from the solution-filled half of the channel into the solvent-filled half at a rate determined by their hydrodynamic radius. The arrival of the solute molecules in the solvent-filled half of the channel is recorded in a spectrally resolved manner by Raman microspectroscopy. From the time series of Raman spectra, a two-dimensional Raman-DOSY spectrum is obtained, which has the Raman frequency on one axis and the diffusion coefficient (or equivalently, hydrodynamic radius) on the other. In this way, Raman-DOSY spectrally resolves overlapping Raman peaks arising from molecules of different sizes. We demonstrate Raman-DOSY on samples containing up to three compounds and derive the diffusion coefficients of small molecules, proteins, and supramolecules (micelles), illustrating the versatility of Raman-DOSY. Raman-DOSY is label-free and does not require deuterated solvents and can thus be applied to samples and matrices that might be difficult to investigate with other diffusion-based spectroscopy methods.

Multidimensional infrared diffusion-ordered spectroscopy in depletion mode distinguishes protein amyloids and monomers.

Conventional and two-dimensional infrared (2D-IR) spectroscopy are well suited to study amyloid aggregates, because the amide I mode is a sensitive probe of the aggregate structure. However, these methods are not so useful to study mixtures of aggregates and monomers, which generally have overlapping amide I spectra. Here, we show that IR-Diffusion-Ordered Spectroscopy can disentangle the contributions of protein monomers and aggregates (amyloids) in FTIR and 2D-IR spectra by separating the spectral contributions based on molecular size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes-Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient inside an IR sample cell and tracking its equilibration in an IR-frequency-resolved manner. The amyloid diffusion is too slow to be experimentally observable, so instead of tracking the arrival of molecular species diffusing into the initially empty region of the sample cell, we track the depletion of the more rapidly diffusing species as they leave the sample-filled region. This way, we can still obtain the spectrum of very slowly diffusing species, although we cannot determine their diffusion coefficient. We first demonstrate this depletion method on a mixture of two small organic molecules and then show how it can be used to separate the spectrum of a mixture of bovine-serum-albumin amyloids and monomers into its component spectra, both in the FTIR and 2D-IR case.

Surface-Mediated Molecular Transport of a Lipophilic Fluorescent Probe in Polydisperse Oil-in-Water Emulsions.

Emulsions often act as carriers for water-insoluble solutes that are delivered to a specific target. The molecular transport of solutes in emulsions can be facilitated by surfactants and is often limited by diffusion through the continuous phase. We here investigate this transport on a molecular scale by using a lipophilic molecular rotor as a proxy for solutes. Using fluorescence lifetime microscopy we track the transport of these molecules from the continuous phase toward the dispersed phase in polydisperse oil-in-water emulsions. We show that this transport comprises two time scales, which vary significantly with droplet size and surfactant concentration, and, depending on the type of surfactant used, can be limited either by transport across the oil-water interface or by diffusion through the continuous phase. By studying the time-resolved fluorescence of the fluorophore, accompanied by molecular dynamics simulations, we demonstrate how the rate of transport observed on a macroscopic scale can be explained in terms of the local environment that the probe molecules are exposed to.

Traces of water catalyze zinc soap crystallization in solvent-exposed oil paints.

The crystallization of metal soaps in polymer matrices is a complex process that affects the stability of oil paintings, as well as the properties of commercial ionomer materials. In the context of conservation of paintings, it is crucial to investigate the influence of solvent exposure on such detrimental chemical processes. Using Fourier transform infrared spectroscopy and a polymer model system that contains metastable amorphous zinc soaps, it is shown that water induces zinc soap crystallization, while solvent swelling alone has no effect. In particular fast-diffusing polar organic solvents with water impurities are able to induce extensive crystallization, delivering high concentrations of water quickly deep into paint layers. Finally, it is demonstrated, both with the model system and real oil paint samples, that even with very short solvent exposure times, significant quantities of crystalline zinc soaps are formed. This strong effect of water impurities in common solvents gives reason to be cautious when conservation treatments are being considered for oil paints that contain zinc white or other water-sensitive chemicals.

Hydrogen Bonds under Stress: Strain-Induced Structural Changes in Polyurethane Revealed by Rheological Two-Dimensional Infrared Spectroscopy.

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.

Infrared Diffusion-Ordered Spectroscopy Reveals Molecular Size and Structure.

Inspired by ideas from NMR, we have developed Infrared Diffusion-Ordered Spectroscopy (IR-DOSY), which simultaneously characterizes molecular structure and size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes-Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient and tracking its equilibration in an IR-frequency resolved manner. Analogous to NMR-DOSY, a two-dimensional IR-DOSY spectrum has IR frequency along one axis and diffusion coefficient (or equivalently, size) along the other, so the chemical structure and the size of a compound are characterized simultaneously. In an IR-DOSY spectrum of a mixture, molecules with different sizes are nicely separated into distinct sets of IR peaks. Extending this idea to higher dimensions, we also perform 3D-IR-DOSY, in which we combine the conformation sensitivity of femtosecond multi-dimensional IR spectroscopy with size sensitivity.

In Situ Identification of Secondary Structures in Unpurified Bombyx mori Silk Fibrils Using Polarized Two-Dimensional Infrared Spectroscopy.

The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behavior of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the noninvasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools because the absorption bands of amide groups strongly depend on protein secondary structure. However, spectral congestion usually complicates the analysis of the amide spectrum. Here, we apply polarized two-dimensional (2D) infrared spectroscopy (IR) to directly identify the protein secondary structures in native silk films cast from Bombyx mori silk feedstock. Without any additional peak fitting, we find that the initial effect of hydration is an increase of the random coil content at the expense of the helical content, while the ß-sheet content is unchanged and only increases at a later stage. This paper demonstrates that 2D-IR can be a valuable tool for characterizing biomaterials.

Broadband Multidimensional Spectroscopy Identifies the Amide II Vibrations in Silkworm Films.

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 ß-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 giant staphylococcal protein Embp facilitates colonization of surfaces through Velcro-like attachment to fibrillated fibronectin.

Staphylococcus epidermidis causes some of the most hard-to-treat clinical infections by forming biofilms: Multicellular communities of bacteria encased in a protective matrix, supporting immune evasion and tolerance against antibiotics. Biofilms occur most commonly on medical implants, and a key event in implant colonization is the robust adherence to the surface, facilitated by interactions between bacterial surface proteins and host matrix components. S. epidermidis is equipped with a giant adhesive protein, extracellular matrix-binding protein (Embp), which facilitates bacterial interactions with surface-deposited, but not soluble fibronectin. The structural basis behind this selective binding process has remained obscure. Using a suite of single-cell and single-molecule analysis techniques, we show that S. epidermidis is capable of such distinction because Embp binds specifically to fibrillated fibronectin on surfaces, while ignoring globular fibronectin in solution. S. epidermidis adherence is critically dependent on multivalent interactions involving 50 fibronectin-binding repeats of Embp. This unusual, Velcro-like interaction proved critical for colonization of surfaces under high flow, making this newly identified attachment mechanism particularly relevant for colonization of intravascular devices, such as prosthetic heart valves or vascular grafts. Other biofilm-forming pathogens, such as Staphylococcus aureus, express homologs of Embp and likely deploy the same mechanism for surface colonization. Our results may open for a novel direction in efforts to combat devastating, biofilm-associated infections, as the development of implant materials that steer the conformation of adsorbed proteins is a much more manageable task than avoiding protein adsorption altogether.

A usually harmless bacterium called Staphylococcus epidermidis lives on human skin. Sometimes it makes its way into the bloodstream through a cut or surgical procedure, but it rarely causes blood infections. It can, however, cause severe infections when it attaches to the surface of a medical implant like a pacemaker or an artificial replacement joint. It does this by forming a colony of bacteria on the implant's surface called a biofilm, which protects the bacteria from destruction by the immune system or antibiotics. Understanding how Staphylococcus epidermidis implant infections start is critical to preventing them. This information may help scientists develop infection-resistant implants or new treatments for implant infections. Scientists suspect that Staphylococcus epidermidis attaches to implants by binding to a human protein called fibronectin, which coats medical implants in the human body. Another protein on the surface of the bacteria, called Embp, facilitates the connection. But why the bacteria attach to fibronectin on implants, and not fibronectin molecules in the bloodstream, is unclear. Now, Khan, Aslan et al. show that Embp forms a Velcro-like bond with fibronectin on the surface of implants. In the experiments, Khan and Aslan et al. used powerful microscopes to create 3-dimensional images of the interactions between Embp and fibronectin. The experiments showed that Embp's attachment site is hidden on the globe-shaped form of fibronectin circulating in the blood. But when fibronectin covers an implant surface, it forms a fibrous network, and Embp can attach to it with up to 50 Velcro-like individual connections. These many weak connections form a strong bond that withstands the force of blood pumping past. The experiments show that the fibrous coating of fibronectin on implants makes them a hotspot for Staphylococcus epidermidis infections. Finding ways to block Embp from attaching to fibronectin on implants, or altering the form fibronectin takes on implants, may help prevent these infections. Many bacteria that form biofilms have an Embp-like protein. As a result, these discoveries may also help scientists develop prevention or treatment strategies for other bacterial biofilm infections.

Fluorescent molecular rotor probes nanosecond viscosity changes.

Viscosity is a key property of liquids, but it is difficult to measure in short-lived, metastable samples due to the long measuring times required by conventional rheology. Here, we show how this problem can be solved by using fluorescent molecular rotors. The excited-state fluorescence decay rate of these molecules is sensitive to the viscosity of their local environment, and by combining pulsed laser excitation with time-resolved fluorescence detection, we can measure viscosities with a time resolution of a few ns. We demonstrate this by measuring in real time the viscosity change in glycerol induced by a nanosecond temperature jump. This new approach makes it possible to measure the viscosity of extremely short-lived states of matter.

Chromatographic separation of active polymer-like worm mixtures by contour length and activity.

The convective transport rate of polymers through confined geometries depends on their size, allowing for size-based separation of polymer mixtures (chromatography). Here, we investigate whether mixtures of active polymers can be separated in a similar manner based on their activity. We use thin, living Tubifex tubifex worms as a model system for active polymers and study the transport of these worms by an imposed flow through a channel filled with a hexagonal pillar array. The transport rate through the channel depends strongly on the degree of activity, an effect that we assign to the different distribution of conformations sampled by the worms depending on their activity. Our results demonstrate a unique way to sort mixtures of active polymers based on their activity and provide a versatile and convenient experimental system to investigate the hydrodynamics of active polymers.

Scratch-Healing Behavior of Ice by Local Sublimation and Condensation.

We show that the surface of ice is scratch healing: micrometer-deep scratches in the ice surface spontaneously disappear by thermal relaxation on the time scale of roughly an hour. Following the dynamics and comparing it to different mass transfer mechanisms, we find that sublimation from and condensation onto the ice surface is the dominant scratch-healing mechanism. The scratch-healing kinetics shows a strong temperature dependence, following an Arrhenius behavior with an activation energy of ΔE = 58.6 ± 4.6 kJ/mol, agreeing with the proposed sublimation mechanism and at odds with surface diffusion or fluid flow or evaporation-condensation from a quasi-liquid layer.

Transition from viscoelastic to fracture-like peeling of pressure-sensitive adhesives.

We investigate the process of the slow unrolling of a roll of typical pressure-sensitive adhesive, Scotch tape, under its own weight. Probing the peeling velocities down to nm s-1 resolution, which is three orders of magnitudes lower than earlier measurements, we find that the speed is still non-zero. Moreover, the velocity is correlated to the relative humidity. A humidity increase leads to water uptake, making the adhesive weaker and easier to peel. At very low humidity, the adhesive becomes so stiff that it mainly responds elastically, leading to a peeling process akin to interfacial fracture. We provide a quantitative understanding of the peeling velocity in the two regimes.

Nonmonotonic Friction due to Water Capillary Adhesion and Hydrogen Bonding at Multiasperity Interfaces.

Capillary adhesion due to water adsorption from the air can contribute to friction, especially for smooth interfaces in humid environments. We show that for multiasperity (naturally oxidized) Si-on-Si interfaces, the friction coefficient goes through a maximum as a function of relative humidity. An adhesion model based on the boundary element method that takes the roughness of the interfaces into account reproduces this nonmonotonic behavior very well. Remarkably, we find the dry friction to be significantly lower than the lubricated friction with macroscopic amounts of water present. The difference is attributed to the hydrogen-bonding network across the interface. Accordingly, the lubricated friction increases significantly if the water is replaced by heavy water (D_{2}O) with stronger hydrogen bonding.

Accelerated Vibrational Energy Relaxation of Water in Alkaline Environments.

We observe that hydrated hydroxide ions introduce an additional relaxation channel for the vibrational relaxation of the OD vibrations of HDO molecules in aqueous NaOH solutions. This additional relaxation path involves resonant (Förster) vibrational energy transfer from the excited OD vibration to OH stretch vibrations of hydrated OH- complexes. This energy transfer constitutes an efficient mechanism for dissipation of the OD vibrational energy, as the accepting OH stretch vibrations show an extremely rapid subsequent relaxation with a time constant of <200 fs. We find that the Förster energy transfer is characterized by a Förster radius of 2.8 ± 0.2 Å.

Correction: Role of additives and solvents in the synthesis of chiral isoreticular MOF-74 topologies.

Correction for 'Role of additives and solvents in the synthesis of chiral isoreticular MOF-74 topologies' by Andreea Gheorghe et al., Dalton Trans., 2021, DOI: 10.1039/D1DT01945G.

Role of additives and solvents in the synthesis of chiral isoreticular MOF-74 topologies.

Chiral induction is a simple and inexpensive approach to synthesise chiral metal-organic frameworks, even when using achiral building-blocks. The challenge lies in selecting the proper chiral inductor. This can only be achieved upon understanding the mechanism behind the chirality transfer from the chiral guest to the achiral MOF. In this work, the role of two types of chiral additives and different solvents was investigated in the crystallization of isoreticular MOF-74. We show that pyrrolidone-based solvents can interact with the framework walls and influence the thermal stability of the MOF. The role of the different chiral additives is related to the strength of their interaction with the MOF. Unlike cinchona alkaloids that have weak interactions with the framework, L- or D-trans-4-hydroxyproline (L- or D-Hyp) can strongly bind to the Zn2+ metal centres and cause the twisting of the organic linker. Moreover, L- and D-Hyp additives can affect the IRMOF-74 nucleation process depending on their concentration and handedness.