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The organic nucleation of the pharmaceutical ibuprofen is investigated, as triggered by the protonation of ibuprofen sodium salt at elevated pH. The growth and aggregation of nanoscale solution species by Analytical Ultracentrifugation and Molecular Dynamics (MD) simulations is tracked. Both approaches reveal solvated molecules, oligomers, and prenucleation clusters, their size as well as their hydration at different reaction stages. By combining surface-specific vibrational spectroscopy and MD simulations, water interacting with ibuprofen at the air-water interface during nucleation is probed. The results show the structure of water changes upon ibuprofen protonation in response to the charge neutralization. Remarkably, the water structure continues to evolve despite the saturation of protonated ibuprofen at the hydrophobic interface. This further water rearrangement is associated with the formation of larger aggregates of ibuprofen molecules at a late prenucleation stage. The nucleation of ibuprofen involves ibuprofen protonation and their hydrophobic assembly. The results highlight that these processes are accompanied by substantial water reorganization. The critical role of water is possibly relevant for organic nucleation in aqueous environments in general., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Studying lipid monolayers as model biological membranes, we demonstrate that water molecules interfacing with different model membranes can display preferential orientation for two distinct reasons: due to charges on the membrane, and due to large dipole fields resulting from zwitterionic headgroups. This preferential water orientation caused by the charge or the dipolar field can be effectively neutralized to net-zero water orientation by introducing monolayer counter-charges ( i.e. lipids with oppositely charged headgroups). Following the Gouy-Chapman model, the effect of monolayer surface charge on water orientation is furthermore strongly dependent on the electrolyte concentration and thus on the counterions in solution. In contrast, the effect of ions in the subphase on the dipolar alignment of water is zero. As a result, the capability of monolayer counter-charges to null the effect of dipolar orientation is strongly electrolyte-dependent. Notably, the different effects are additive for mixed charged/zwitterionic lipid systems occurring in nature. Specifically, for an E. coli lipid membrane extract consisting of both zwitterionic and negatively charged lipids, the water orientation can be explained by the sum of the constituents. Our results can be quantitatively reproduced using Gouy-Chapman theory, revealing the relatively straightforward electrostatic effects on the hydration of complex membrane interfaces.

Understanding the nucleation of iron oxides and the underlying hydrolysis of aqueous iron species is still challenging, and molecular-level insights into the orchestrated response of water, especially at the hydrolysis interface, are lacking. We follow iron(III) hydrolysis in the presence of a synthetic bacterial iron nucleator, which is a magnetosome membrane specific peptide, by using a constant pH titration technique. Three distinct hydrolysis regimes were identified. Interface-selective sum frequency generation (SFG) spectroscopy was used to probe the interfacial reaction and water in direct contact with the peptide. SFG data reveal that iron(III) species react quickly with interfacial peptides while continuously enhancing water alignment into the later stages of hydrolysis. The gradually aligning water molecules are associated with initially promoted (regimes I and II) and later suppressed (regime III) hydrolysis after the saturation of water alignment has occurred until regime II. These interfacial insights are crucial for understanding the early stage of iron oxide biomineralization.

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum . We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits. Fusarium INs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs.

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.

Accurate determination of protein structure at interfaces is critical for understanding protein interactions, which is directly relevant to a molecular-level understanding of interfacial proteins in biology and medicine. Vibrational sum frequency generation (VSFG) spectroscopy is often used for probing the protein amide I mode, which reports protein structures at interfaces. Observed peak shifts are attributed to conformational changes and often form the foundation of hypotheses explaining protein working mechanisms. Here, we investigate structurally diverse proteins using conventional and heterodyne-detected VSFG (HD-VSFG) spectroscopy as a function of solution pH. We reveal that blue-shifts of the amide I peak observed in conventional VSFG spectra upon lowering the pH are governed by the drastic change of the nonresonant contribution. Our results highlight that connecting changes in conventional VSFG spectra to conformational changes of interfacial proteins can be arbitrary, and that HD-VSFG measurements are required to draw unambiguous conclusions about structural changes in biomolecules.

Understanding the collective behavior of ions at charged surfaces is of paramount importance for geological and electrochemical processes. Ions screen the surface charge, and interfacial fields break the centro-symmetry near the surface, which can be probed using second-order nonlinear spectroscopies. The effect of electrolyte concentration on the nonlinear optical response has been semi-quantitatively explained by mean-field models based on the Poisson-Boltzmann equation. Yet, to explain previously reported ion-specific effects on the spectroscopic response, drastic ion-specific changes in the interfacial properties, including surface acidities and dielectric permittivities, or strong ion adsorption/desorption had to be invoked. Here, we use sum-frequency generation (SFG) spectroscopy to probe the symmetry-breaking of water molecules at a charged silica surface in contact with alkaline metal chloride solutions (LiCl, NaCl, KCl, and CsCl) at various concentrations. We find that the water response varies with the cation: the SFG response is markedly enhanced for LiCl compared to CsCl. We show that within mean-field models, neither specific ion-surface interactions nor a reduced dielectric constant of water near the interface can account for the variation of spectral intensities with cation nature. Molecular dynamics simulations confirm that the decay of the electrochemical potential only weakly depends on the salt type. Instead, the effect of different salts on the optical response is indirect, through the reorganization of the interfacial water: the salt-type-dependent alignment of water directly at the interface can explain the observations.

Assembly by amyloid-beta (Aβ) peptides is vital for various neurodegenerative diseases. The process can be accelerated by hydrophobic interfaces such as the cell membrane interface and the air-water interface. Elucidating the assembly mechanism for Aβ peptides at hydrophobic interface requires knowledge of the microscopic structure of interfacial peptides. Here we combine scanning force microscopy, sum-frequency generation spectroscopy, and metadynamics simulations to probe the structure of the central fragment of Aβ peptides at the air-water interface. We find that the structure of interfacial peptides depends on pH: at neutral pH, the peptides adopt a less folded, bending motif by forming intra-hydrogen bonds; at acidic pH, the peptides refold into extended β-strand fibril conformation, which further promotes their macroscopic assembly. The conformational transition of interfacial peptides is driven by the reduced hydrogen bonds, both with water and within peptides, resulting from the protonation of acidic glutamic acid side chains.

The three-dimensional spatial distribution of molecules at soft matter interfaces is crucial for processes ranging from membrane biophysics to atmospheric chemistry. While several techniques can access surface composition, obtaining information on the depth distribution is challenging. We develop a noninvasive, polarization-resolved, surface-specific sum-frequency generation spectroscopy providing quantitative depth information. We demonstrate the technique on formic acid molecules at the air-water interface. With increasing molar fraction from 2.5% to 10%, the formic acid molecules shift, on average, ∼0.9  Å into the bulk. The consistency with the simulation data manifests that the technique allows for probing the Ångstrom-scale depth profile.

Bacterial ice nucleators (INs) are among the most effective ice nucleators known and are relevant for freezing processes in agriculture, the atmosphere, and the biosphere. Their ability to facilitate ice formation is due to specialized ice-nucleating proteins (INPs) anchored to the outer bacterial cell membrane, enabling the crystallization of water at temperatures up to -2 °C. In this Perspective, we highlight the importance of functional aggregation of INPs for the exceptionally high ice nucleation activity of bacterial ice nucleators. We emphasize that the bacterial cell membrane, as well as environmental conditions, is crucial for a precise functional INP aggregation. Interdisciplinary approaches combining high-throughput droplet freezing assays with advanced physicochemical tools and protein biochemistry are needed to link changes in protein structure or protein-water interactions with changes on the functional level.

The origin of the sum-frequency generation (SFG) signal of the water bending mode has been controversially debated in the past decade. Unveiling the origin of the signal is essential, because different assignments lead to different views on the molecular structure of interfacial water. Here, we combine collinear heterodyne-detected SFG spectroscopy at the water-charged lipid interfaces with systematic variation of the salt concentration. The results show that the bending mode response is of a dipolar, rather than a quadrupolar, nature and allows us to disentangle the response of water in the Stern and the diffuse layers. While the diffuse layer response is identical for the oppositely charged surfaces, the Stern layer responses reflect interfacial hydrogen bonding. Our findings thus corroborate that the water bending mode signal is a suitable probe for the structure of interfacial water.

Biomineralization occurs in aqueous environments. Despite the ubiquity and relevance of CaCO 3 biomineralization, the role of water in the biomineralization process has remained elusive. Here, we demonstrate that water reorganization accompanies CaCO 3 biomineralization for sea urchin spine generation in a model system. Using surface-specific vibrational spectroscopy, we probe the water at the interface of the spine-associated protein during CaCO 3 mineralization. Our results show that, while the protein structure remains unchanged, the structure of interfacial water is perturbed differently in the presence of both Ca 2+ and CO 3 2- compared to the addition of only Ca 2+ . This difference is attributed to the condensation of prenucleation mineral species. Our findings are consistent with a nonclassical mineralization pathway for sea urchin spine generation and highlight the importance of protein hydration in biomineralization.

Ice-nucleating proteins (INPs) found in bacteria are the most effective ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C. Although their function has been known for decades, the underlying mechanism is still under debate. Here, we show that INPs from Pseudomonas syringae in aqueous solution exhibit a defined solution structure and show no significant conformational changes upon cooling. In contrast, irreversible structural changes are observed upon heating to temperatures exceeding ∼55 °C, leading to a loss of the ice-nucleation activity. Sum-frequency generation (SFG) spectroscopy reveals that active and heat-inactivated INPs impose similar structural ordering of interfacial water molecules upon cooling. Our results demonstrate that increased water ordering is not sufficient to explain INPs' high ice-nucleation activity and confirm that intact three-dimensional protein structures are critical for bacterial ice nucleation, supporting a mechanism that depends on the INPs' supramolecular interactions.

The structure of interfacial water determines atmospheric chemistry, wetting properties of materials, and protein folding. The challenge of investigating the properties of specific interfacial water molecules has frequently been confronted using surface-specific sum-frequency generation (SFG) vibrational spectroscopy using the O-H stretch mode. While perfectly suited for the water-air interface, for complex interfaces, a potential complication arises from the contribution of hydroxyl or amine groups of non-water species present at the surface, such as surface hydroxyls on minerals, or O-H and N-H groups contained in proteins. Here, we present a protocol to extract the hydrogen bond strength selectively of interfacial water, through the water bending mode. The bending mode vibrational frequency distribution provides a new avenue for unveiling the hydrogen bonding structure of interfacial water at complex aqueous interfaces. We demonstrate this method for the water-CaF 2 and water-protein interfaces. For the former, we show that this method can indeed single out water O-H groups from surface hydroxyls, and that with increasing pH, the hydrogen-bonded network of interfacial water strengthens. Furthermore, we unveil enhanced hydrogen bonding of water, compared to bulk water, at the interface with human serum albumin proteins, a prototypical bio-interface.

The structure of water molecules in contact with zwitterionic lipid molecules is of great biological relevance, because biological membranes are largely composed of such lipids. The interaction of the interfacial water molecules with the amphiphilic lipid molecules drives the formation of membranes and greatly influences various processes at the membrane surface, as the field that arises from the aligned interfacial water molecules masks the charges of the lipid headgroups from the approaching metabolites. To increase our understanding of the influence of water molecules on biological processes we study their structure at the interface using sum-frequency generation spectroscopy and molecular dynamics simulations. Interestingly, we find that water molecules at zwitterionic lipid molecules are mainly oriented by the field arising between the two oppositely charged molecular moieties within the lipid headgroups.

The electrical charge of biological membranes and thus the resulting alignment of water molecules in response to this charge are important factors affecting membrane rigidity, transport, and reactivity. We tune the surface charge density by varying lipid composition and investigate the charge-induced alignment of water molecules using surface-specific vibrational spectroscopy and molecular dynamics simulations. At low charge densities, the alignment of water increases proportionally to the charge. However, already at moderate, physiologically relevant charge densities, water alignment starts to saturate despite the increase in the nominal surface charge. The saturation occurs in both the Stern layer, directly at the surface, and in the diffuse layer, yet for distinctly different reasons. Our results show that the soft nature of the lipid interface allows for a marked reduction of the surface potential at high surface charge density via both interfacial molecular rearrangement and permeation of monovalent ions into the interface.

Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, and to a lesser extent to other biomolecules and ions.The cytoplasm's crowdedness leads one to expect that cell water is different from bulk water. By measuring the rotational motion of water molecules in living cells, Tros et al. find that apart from a small fraction of water solvating biomolecules, cell water has the same dynamics as bulk water.

Understanding aqueous interfaces at the molecular level is not only fundamentally important, but also highly relevant for a variety of disciplines. For instance, electrode-water interfaces are relevant for electrochemistry, as are mineral-water interfaces for geochemistry and air-water interfaces for environmental chemistry; water-lipid interfaces constitute the boundaries of the cell membrane, and are thus relevant for biochemistry. One of the major challenges in these fields is to link macroscopic properties such as interfacial reactivity, solubility, and permeability as well as macroscopic thermodynamic and spectroscopic observables to the structure, structural changes, and dynamics of molecules at these interfaces. Simulations, by themselves, or in conjunction with appropriate experiments, can provide such molecular-level insights into aqueous interfaces. In this contribution, we review the current state-of-the-art of three levels of molecular dynamics (MD) simulation: ab initio, force field, and coarse-grained. We discuss the advantages, the potential, and the limitations of each approach for studying aqueous interfaces, by assessing computations of the sum-frequency generation spectra and surface tension. The comparison of experimental and simulation data provides information on the challenges of future MD simulations, such as improving the force field models and the van der Waals corrections in ab initio MD simulations. Once good agreement between experimental observables and simulation can be established, the simulation can be used to provide insights into the processes at a level of detail that is generally inaccessible to experiments. As an example we discuss the mechanism of the evaporation of water. We finish by presenting an outlook outlining four future challenges for molecular dynamics simulations of aqueous interfacial systems.

We present a combined experimental sum-frequency generation (SFG) spectroscopy and ab initio molecular dynamics simulations study to clarify the structure and orientation of water at zwitterionic phosphatidylcholine (PC) lipid and amine N-oxide (AO) surfactant monolayers. Simulated O-H stretch SFG spectra of water show good agreement with the experimental data. The SFG response at the PC interface exhibits positive peaks, whereas both negative and positive bands are present for the similar zwitterionic AO interface. The positive peaks at the water/PC interface are attributed to water interacting with the lipid carbonyl groups, which act as efficient hydrogen bond acceptors. This allows the water hydrogen bond network to reach, with its (up-oriented) O-H groups, into the headgroup of the lipid, a mechanism not available for water underneath the AO surfactant. This highlights the role of the lipid carbonyl group in the interfacial water structure at the membrane interface, namely, stabilizing the water hydrogen bond network.

The amphiphilic osmolyte trimethylamine-N-oxide (TMAO) is commonly found in natural organisms, where it counteracts biochemical stress associated with urea in aqueous environments. Despite the important role of TMAO as osmoprotectant, the mechanism behind TMAO's action has remained elusive. Here, we study the interaction between urea, TMAO, and water in solution using broadband (100 MHz-1.6 THz) dielectric spectroscopy. We find that the previously reported tight hydrogen bonds between 3 water molecules and the hydrophilic amine oxide group of TMAO, remain intact at all investigated concentrations of urea, showing that no significant hydrogen bonding occurs between the two co-solutes. Despite the absence of direct TMAO-urea interactions, the solute reorientation times of urea and TMAO show an anomalous nonlinear increase with concentration, for ternary mixtures containing equal amounts of TMAO and urea. The nonlinear increase of the reorientation correlates with changes in the viscosity, showing that the combination of TMAO and urea cooperatively enhances the hydrogen-bond structure of the ternary solutions. This nonlinear increase is indicative of water mediated interaction between the two solutes and is not observed if urea is combined with other amphiphilic solutes.

GALA is a 30 amino acid synthetic peptide consisting of a Glu-Ala-Leu-Ala repeat and is known to undergo a reversible structural transition from a disordered to an α-helical structure when changing the pH from basic to acidic values. In its helical state GALA can insert into and disintegrate lipid membranes. This effect has generated much interest in GALA as a candidate for pH triggered, targeted drug delivery. GALA also serves as a well-defined model system to understand cell penetration mechanisms and protein folding triggered by external stimuli. Structural transitions of GALA in solution have been studied extensively. However, cell penetration is an interfacial effect and potential biomedical applications of GALA would involve a variety of surfaces, e.g., nanoparticles, lipid membranes, tubing, and liquid-gas interfaces. Despite the apparent importance of interfaces in the functioning of GALA, the effect of surfaces on the reversible folding of GALA has not yet been studied. Here, we use sum frequency generation vibrational spectroscopy (SFG) to probe the structural response of GALA at the air-water interface and IR spectroscopy to follow GALA folding in bulk solution. We combine the SFG data with molecular dynamics simulations to obtain a molecular-level picture of the interaction of GALA with the air-water interface. Surprisingly, while the fully reversible structural transition was observed in solution, at the water-air interface, a large fraction of the GALA population remained helical at high pH. This "stickiness" of the air-water interface can be explained by the stabilizing interactions of hydrophobic leucine and alanine side chains with the water surface.

We report molecular dynamics (MD) simulations of the water/clean rutile TiO2 (110) interface using polarizable and non-surface polarity force field models. The effect of surface polarity on the water dynamics near the TiO2(110) surface is addressed, specifically by calculating the water hydrogen bond and reorientational dynamics. The hydrogen bond lifetime of interfacial water molecules is several times longer than that of bulk water due to the strong water-TiO2 interactions. A comparison of the dynamics simulated with the polarizable and non-surface polarity models shows that, while the hydrogen bond lifetime between the interfacial water and TiO2 surface is insensitive to the surface polarity, the reorientational dynamics around this hydrogen bond axis is significantly influenced by the surface polarity; the surface polarity of the TiO2 increases the water-TiO2 interactions, stabilizing the local structure of the interfacial water molecules and restricting their rotational motion. This reorientation occurs predominantly by rotation around the O-H group hydrogen bonded to the TiO2 surface. Furthermore, we correlate the dynamics of the induced charge on the TiO2 surface with the interfacial water dynamics. Our results show that the timescale of correlations of the atom charges induced by the local electric field in bulk water is influenced by the rotational motion, hydrogen bond rearrangement and translational motion, while the induced charge dynamics of the TiO2 surface is governed primarily by the rotational dynamics of the interfacial water molecules. This study demonstrates that the solid surface polarity has a significant impact on the dynamics of water molecules near TiO2 surfaces.

In this work, we present evidence for ion pair formation of cations with a high surface charge density (Na(+) and Ca(2+)) and phosphate groups of phospholipids. We used femto-second infrared pump-probe and dielectric spectroscopy to probe the dynamics of water molecules in solutions of phosphorylethanolamine and different types of cations. We find that sodium and calcium cooperatively retard the dynamics of water in solutions of phosphorylethanolamine, implying the formation of solvent separated ion pairs. This ion-specific interaction is absent for potassium, cesium and ammonium. We compare our results to dielectric spectroscopy experiments, which probes the rotation of all dipolar molecules and ions in solution. The rotation of the dipolar phosphorylethanolamine ion shows that long-lived ion-pairs are only formed with calcium and not with ammonium, cesium, potassium, and sodium. This finding implies that the association between calcium and the phosphate is strong with lifetimes exceeding 200 ps, while the interaction with sodium is relatively short-lived (∼20-100 ps).

Interfaces of liquid water play a critical role in a wide variety of processes that occur in biology, a variety of technologies, and the environment. Many macroscopic observations clarify that the properties of liquid water interfaces significantly differ from those of the bulk liquid. In addition to interfacial molecular structure, knowledge of the rates and mechanisms of the relaxation of excess vibrational energy is indispensable to fully understand physical and chemical processes of water and aqueous solutions, such as chemical reaction rates and pathways, proton transfer, and hydrogen bond dynamics. Here we elucidate the rate and mechanism of vibrational energy dissipation of water molecules at the air-water interface using femtosecond two-color IR-pump/vibrational sum-frequency probe spectroscopy. Vibrational relaxation of nonhydrogen-bonded OH groups occurs at a subpicosecond timescale in a manner fundamentally different from hydrogen-bonded OH groups in bulk, through two competing mechanisms: intramolecular energy transfer and ultrafast reorientational motion that leads to free OH groups becoming hydrogen bonded. Both pathways effectively lead to the transfer of the excited vibrational modes from free to hydrogen-bonded OH groups, from which relaxation readily occurs. Of the overall relaxation rate of interfacial free OH groups at the air-H2O interface, two-thirds are accounted for by intramolecular energy transfer, whereas the remaining one-third is dominated by the reorientational motion. These findings not only shed light on vibrational energy dynamics of interfacial water, but also contribute to our understanding of the impact of structural and vibrational dynamics on the vibrational sum-frequency line shapes of aqueous interfaces.

The surface vibrational spectrum of water at biological interfaces is often interpreted as having 'ice-like' and 'liquid-like' components. Here we show that the vibrational spectrum of water at both water-lipid and water-protein interfaces greatly simplifies upon H/D isotopic dilution, which is inconsistent with the presence of 'ice-like' structures. The changes in the spectra as a function of isotope content can be explained by intramolecular coupling between bend and stretch vibrations of the water molecules.

We report the complete assignment of the vibrational spectrum of dipalmitoylphosphatidylcholine (DPPC), which belongs to the most ubiquitous membrane phospholipid family, phosphatidylcholine. We find that water hydrating the lipid headgroups enables efficient energy transfer across membrane leaflets on sub-picosecond time scales. The emergence of spatially extended vibrational modes upon hydration, underlies this phenomenon. Our findings illustrate the importance of collective molecular behavior of biomembranes and reveal that hydrated lipid membranes can act as efficient media for the transfer of vibrational energy.

We report a combined vibrational sum-frequency generation (SFG) spectroscopy, Brewster angle microscopy (BAM), and ellipsometry study of different surfactants on water as a function of surfactant density. Vibrational SFG spectra of surfactants on the water surface in a Langmuir trough have been measured in both the surfactant CH and the water OH stretch regions. At low densities, the SFG signal generated at the surface in the presence of the surfactant is indistinguishable from the SFG signal generated at the clean water-air interface. When the surfactant density increases, i.e., upon compressing the monolayer, a very sudden increase in the SFG signal in both the CH and OH spectral regions is observed. For higher laser fluences, this stepwise increase occurs at increasingly higher surfactant densities. Since BAM shows that surfactant molecules are clearly present at these low densities, we conclude that at low surfactant density the laser beam displaces relatively high-density domains with surfactants in the liquid expanded phase out of the region of the laser focus. This is a consequence of the thermal gradient induced by local heating of the water phase with the monolayer on top due to repetitive laser excitation at 1 kHz. It can be circumvented by using a rotating trough. In this manner, the sampled surface area can be refreshed, allowing artifact-free vibrational SFG spectra to be measured down to the very lowest surfactant densities. In ellipsometry experiments, a similar step can be noticed, which, however, is of a different nature; i.e., it is not related to heating (the laser fluence is very low and the light nonresonant) but to a molecular transition. The occurrence of the step in ellipsometry as a function of area per molecule depends critically on the preparation of the monolayer. By giving the molecules time and space to relax during the preparation of the monolayer, this step could also be eliminated.

Water is very different from liquids of similar molecular weight, and one of its unique properties is the very efficient transfer of vibrational energy between molecules, which arises as a result of strong dipole-dipole interactions between the O-H oscillators. Although we have a sound understanding of such energy transfer in bulk water, we know less about how, and how quickly, transfer occurs at its interface with a hydrophobic phase, because specifically addressing the outermost monolayer is difficult. Here, we use ultrafast two-dimensional surface-specific vibrational spectroscopy to probe the interfacial energy dynamics of heavy water (D(2)O) at the water/air interface. The measurements reveal the presence of surprisingly rapid energy transfer, both between hydrogen-bonded interfacial water molecules (intermolecular), and between O-D groups sticking out from the water surface and those located on the same molecule and pointing towards the water bulk (intramolecular). Vibrational energy transfer occurs on sub-picosecond timescales, and its rates and pathways can be quantified directly.

We report the vibrational and orientational dynamics of water molecules in isotopically diluted NaOH and NaOD solutions using polarization-resolved femtosecond vibrational spectroscopy and terahertz time-domain dielectric relaxation measurements. We observe a speed-up of the vibrational relaxation of the O-D stretching vibration of HDO molecules outside the first hydration shell of OH(-) from 1.7 ± 0.2 ps for neat water to 1.0 ± 0.2 ps for a solution of 5 M NaOH in HDO:H(2)O. For the O-H vibration of HDO molecules outside the first hydration shell of OD(-), we observe a similar speed-up from 750 ± 50 fs to 600 ± 50 fs for a solution of 6 M NaOD in HDO:D(2)O. The acceleration of the decay is assigned to fluctuations in the energy levels of the HDO molecules due to charge transfer events and charge fluctuations. The reorientation dynamics of water molecules outside the first hydration shell are observed to show the same time constant of 2.5 ± 0.2 ps as in bulk liquid water, indicating that there is no long range effect of the hydroxide ion on the hydrogen-bond structure of liquid water. The terahertz dielectric relaxation experiments show that the transfer of the hydroxide ion through liquid water involves the simultaneous motion of ~7 surrounding water molecules, considerably less than previously reported for the proton., (© 2011 American Institute of Physics)

We report the real-time measurement of the ultrafast reorientational motion of water molecules at the water-air interface, using femtosecond time- and polarization-resolved vibrational sum-frequency spectroscopy. Vibrational excitation of dangling OH bonds along a specific polarization axis induces a transient anisotropy that decays due to the reorientation of vibrationally excited OH groups. The reorientation of interfacial water is shown to occur on subpicosecond time scales, several times faster than in the bulk, which can be attributed to the lower degree of hydrogen bond coordination at the interface. Molecular dynamics simulations of interfacial water dynamics are in quantitative agreement with experimental observations and show that, unlike in bulk, the interfacial reorientation occurs in a largely diffusive manner.

We have obtained molecular insights into a monolayer of azobenzene-based photoswitchable lipids self-assembled on water, using the surface sensitive technique vibrational sum-frequency generation spectroscopy in combination with surface pressure measurements. The photolipids can undergo wavelength-dependent, light-triggered cis/trans and trans/cis isomerization, allowing for reversible control of the surface pressure and the molecular ordering of the lipids in the monolayer. If the photoswitchable lipid is embedded in a layer with conventional phospholipids, such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), we show that the surface pressure and molecular ordering of DPPC can be influenced by switching the azobenzene-based lipid between its two states. Remarkably, the state with the higher surface pressure (cis-state) is characterized by a lower degree of molecular order. This counterintuitive result can be understood by noting that the azobenzene moiety in the cis state has a higher dipole moment and therefore favors interaction with water. The surface free energy of the system is lowered (increase of surface pressure) by electrostatic interactions with the lipid headgroups at the interface, resulting in a loop formation of the lipid tail with the cis-azobenzene. This disorder in the tail of the photoswitchable lipid perturbs as well the ordering of DPPC., (© 2011 American Chemical Society)

We study the influence of the amphipilic compound tetramethylurea (TMU) on the dynamical properties of water, using dielectric relaxation spectroscopy in the regime between 0.2 GHz and 2 THz. This technique is capable of resolving different water species, their relative fractions, and their corresponding reorientation dynamics. We find that the reorientation dynamics of water molecules in the hydration shell of the hydrophobic groups of TMU is between 3 (at low concentrations) and 10 (at higher concentrations) times slower than the dynamics of bulk water. The data indicate that the effect of hydrophobic groups on water is strong but relatively short-ranged. With increasing temperature, the fraction of water contained in the hydrophobic hydration shell decreases, which implies that the overall effect of hydrophobic groups on water becomes smaller.

Duramycin is a small tetracyclic peptide which binds specifically to ethanolamine phospholipids (PE). In this study, we used lipid monolayers consisting of 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) and various phosphatidylcholines (PC) to investigate the effect of duramycin on the organization of lipids and its influence on surrounding water molecules, using vibrational sum-frequency generation spectroscopy in conjunction with surface pressure measurements and fluorescence microscopy. The results show that while duramycin has no effect on the PC lipid monolayers, it induces significant disorder of PE molecules and causes an increase of the PE monolayer surface pressure. Duramycin adopts a β-sheet conformation and is well-ordered at the air-water interface as well as after binding to PE. Our results are consistent with duramycin inserting into the PE monolayer via its hydrophobic end, exposing phenylalanine residues to the lipid. Binding of duramycin to PE broadens the hydrogen-bond distribution of lipid-bound water molecules, notably increasing the fraction of the less strongly hydrogen-bonded, possibly undercoordinated, water molecules. Fluorescence microscopy reveals that the interaction of duramycin with PE causes a change in the shape of the liquid-condensed domains of the PE monolayer from circular to horseshoe-like, indicating a reduction of line tension at the boundary of the two lipid phases. These results reveal that the first steps in the disruption of membrane integrity by duramycin consist of a reduction of the line tension, a decrease in the lipid order, and a weakening of the hydrogen bonding network of water around PE.

Understanding the molecular mechanism of DNA/lipid interaction is critical in optimizing the use of lipid cofactors in gene therapy. Here, we address this question by employing label-free vibrational sum frequency (VSF) spectroscopy to study the interaction of DNA with lipid monolayers of the cationic lipids DPTAP(1,2-dipalmitoyl-3-trimethylammonium-propane) and diC14-amidine as well as the zwitterionic lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) in the presence and absence of calcium. Our approach has the advantage both of allowing us to explicitly probe intermolecular interactions and of providing insight into the structure of water and lipids around DNA at the lipid interface. We find, by examination of the OD stretch of interfacial D(2)O, that water structure differs markedly between systems containing DNA adsorbed to cationic and those that contain DNA adsorbed to zwitterionic lipid monolayers (in the presence or absence of Ca(2+)). The spectral response of interfacial water in the cationic system is consistent with a highly structured, undercoordinated, structural 'type' of water. Further, by investigation of CH stretch modes of the diC14-amidine lipid tails, we demonstrate that the adsorption of DNA to this lipid leads to increased ordering of lipid tails.

Fluorescence microscopy and conductivity measurements reveal a remarkably strong effect of hydrophobic groups on the mobility of protons in water. The addition of 5 M of tetramethylurea (4 methyl groups per molecule) results in a reduction of the proton mobility by a factor of approximately 10: hydrophobic hydration strongly suppresses proton mobility. These observations demonstrate the collective nature of aqueous proton transport.

We investigate the structure and orientation of water molecules at the water-lipid interface, using vibrational sum-frequency generation in conjunction with a maximum entropy phase retrieval method. We find that interfacial water molecules have an orientation opposite to that predicted by electrostatics and thus are likely localized between the lipid headgroup and its apolar alkyl chain. This type of water molecule is observed for phospholipids but not for structurally simpler surfactants.

The last decade has seen a transformation in understanding of the role of membrane-bound interfacial water. Whereas until recently water was treated principally as a continuum (primarily screening charges of lipids and proteins), it has become apparent recently that consideration of water's molecular-level properties is critical in understanding a variety of biochemical and biophysical processes. Here we investigate the structure and dynamics of water in contact with a monolayer of artificial lung surfactant, composed of four types of lipids and one protein. We probe this water using frequency-domain sum-frequency generation (SFG) spectroscopy, and a newly developed time-domain, three-pulse technique, in which the vibrational relaxation of interfacial water molecules is followed in real time. We characterize interfacial water in three systems: a monolayer of the pure lipid that is the majority of the lung surfactant mixture, a monolayer of the four lipids constituting the mixture, and a monolayer of the four lipids and the protein. We find subtle differences in the water structure and dynamics that depend on the mixture density and composition. In particular, frequency-domain measurements suggest that in the lipid mixture and the lipid mixture + protein, the relatively bulky lipids (those that have either three or unsaturated hydrocarbon tails) tend to be squeezed out at higher pressure. Measurements using the time-domain, three-pulse technique make clear that structural relaxation of interfacial water is significantly slowed down upon adding small amounts of protein to the lipids. Both results are consistent with prior measurements using other techniques in which more fluid lipids were shown to be 'squeezed out' of lung surfactant at high compression and the role of protein in the mixture was demonstrated to be a catalyst for the formation of multilayers under compression that are subsequently reintegrated into the monolayer on expansion.

Using the surface-specific vibrational technique of vibrational sum-frequency generation, we reveal that the double-peaked structure in the vibrational spectrum of hydrogen-bonded interfacial water molecules originates from vibrational coupling between the stretch and bending overtone, rather than from structural effects. This is demonstrated by isotopic dilution experiments, which reveal a smooth transition from two peaks to one peak, as D2O is converted into HDO. Our results show that the water interface is structurally more homogeneous than previously thought.

We report a femtosecond time-resolved study of water at the neat water-air interface. The O-H stretch vibrational lifetime of hydrogen-bonded interfacial water is measured using surface-specific 4th-order nonlinear optical spectroscopy with femtosecond infrared pulses. The vibrational lifetime in the frequency range of 3200 to 3500 cm(-1) is found to closely resemble that of bulk water, indicating ultrafast exchange of vibrational energy between surface water molecules and those in the bulk.

On the surface of water ice, a quasi-liquid layer (QLL) has been extensively reported at temperatures below its bulk melting point at 273 K. Approaching the bulk melting temperature from below, the thickness of the QLL is known to increase. To elucidate the precise temperature variation of the QLL, and its nature, we investigate the surface melting of hexagonal ice by combining noncontact, surface-specific vibrational sum frequency generation (SFG) spectroscopy and spectra calculated from molecular dynamics simulations. Using SFG, we probe the outermost water layers of distinct single crystalline ice faces at different temperatures. For the basal face, a stepwise, sudden weakening of the hydrogen-bonded structure of the outermost water layers occurs at 257 K. The spectral calculations from the molecular dynamics simulations reproduce the experimental findings; this allows us to interpret our experimental findings in terms of a stepwise change from one to two molten bilayers at the transition temperature.

Interfacial water structures have been studied intensively by probing the O-H stretch mode of water molecules using sum-frequency generation (SFG) spectroscopy. This surface-specific technique is finding increasingly widespread use, and accordingly, computational approaches to calculate SFG spectra using molecular dynamics (MD) trajectories of interfacial water molecules have been developed and employed to correlate specific spectral signatures with distinct interfacial water structures. Such simulations typically require relatively long (several nanoseconds) MD trajectories to allow reliable calculation of the SFG response functions through the dipole moment-polarizability time correlation function. These long trajectories limit the use of computationally expensive MD techniques such as ab initio MD and centroid MD simulations. Here, we present an efficient algorithm determining the SFG response from the surface-specific velocity-velocity correlation function (ssVVCF). This ssVVCF formalism allows us to calculate SFG spectra using a MD trajectory of only ~100 ps, resulting in the substantial reduction of the computational costs, by almost an order of magnitude. We demonstrate that the O-H stretch SFG spectra at the water-air interface calculated by using the ssVVCF formalism well reproduce those calculated by using the dipole moment-polarizability time correlation function. Furthermore, we applied this ssVVCF technique for computing the SFG spectra from the ab initio MD trajectories with various density functionals. We report that the SFG responses computed from both ab initio MD simulations and MD simulations with an ab initio based force field model do not show a positive feature in its imaginary component at 3100 cm-1. [ABSTRACT FROM AUTHOR]

Water must be effectively transported and is also essential for maximizing proton conductivity within fuel-cell proton-exchange membranes (PEMs). Therefore, identifying relationships between PEM properties, water transport, and proton conductivity is essential for designing optimal PEMs. Here, we use coherent Raman spectroscopy to quantify real-time, in situ diffusivities of water subspecies, bulk-like and nonbulk-like (interfacial) water, in five different perfluorosulfonic acid (PFSA) PEMs. Although the PEMs were chemically diverse, water transport within them followed the same rule: Total water diffusivity could be represented by a linear combination of the bulk-like and interfacial water diffusivities. Moreover, the diffusivity of interfacial water was consistently larger than that of bulk-like water. These measurements of microscopic transport were combined with through-plane proton conductivity measurements to reveal the correlation between interfacial water transport and proton conductivity. Our results demonstrate the importance of maximizing the diffusivity and fractional contribution of interfacial water to maximize the proton conductivity in PFSA PEMs. [ABSTRACT FROM AUTHOR]

Knowledge of the interfacial water structure is essential for a basic understanding of the many environmental, technological, and biophysical systems in which aqueous interfaces appear. Using ultrafast two-dimensional surface-specific vibrational spectroscopy we show that the structure of heavy water at the water-air interface displays short-lived heterogeneity and is very different from that at the water-lipid interface. [ABSTRACT FROM AUTHOR]

Abstract: Membranes are highly complex and heterogeneous interfaces that are the active partition between living cells and the outside world. Many biologically important processes occur at the membrane surface, such as transmembrane transport and signaling. Many of these processes depend on the subtle interactions between the different membrane constituents: lipids, proteins and water. At present, a large body of knowledge exists on the molecular composition and static structure of membranes. However, our understanding of the dynamics of membrane molecules has not yet reached the same level of sophistication. Information on membrane dynamics, such as conformational fluctuations, conformational changes and dynamical interactions between membrane constituents are essential for a full understanding of membrane action. Here, we review a recently developed approach aimed at obtaining such dynamical information. The approach is based on surface-specific femtosecond laser vibrational spectroscopy, and is illustrated for simple membrane model systems. [Copyright &y& Elsevier]

We elucidate the structure of interfacial water molecules at the surface of solid ice, using surface-specific vibrational spectroscopy of interfacial water molecules. While in the bulk of ice, each water molecule is fourfold coordinated, both accepting and donating two hydrogen bonds, the hydrogen-bonded network is abruptly interrupted at the surface. The resulting under-coordination of ice molecules at the interface determines many of the properties of the ice surface. Using single-crystal ice, under well-defined conditions, we study the temperature dependence of the outermost surface monolayer of water molecules. We find an excess of hydrogen bonds at the ice-vapor interface around 200 K, due to a competition between entropic and enthalpic contributions to the free energy. At higher temperatures, surface melting of ice is found to occur in a bilayer-by-bilayer fashion. Finally, we show that the temperature-dependent interfacial molecular structure correlates with the temperature dependent macroscopic friction coefficient of ice. [ABSTRACT FROM AUTHOR]