Molecular ordering effects in bulk and at lipid/water nanoscopic interfaces: A story of water, light, and order

Water is ubiquitous on Earth, playing a critical role for a plethora of structures, processes and chemical reactions in nature. Its unique physicochemical properties originate mainly from its hydrogen bond network. Despite its recognized importance, many aspects of the structure of water as a liquid remain elusive. It is especially difficult to understand the complex nature of fluctuations and ordering in pure water, or to get a complete picture of aqueous interfaces at the molecular level. In this thesis, our aim is to improve our understanding of the underlying interactions between water and ions, macromolecules, and interfaces, especially the long range interactions. To this aim, we use two nonlinear optical techniques, second harmonic scattering (SHS) and vibrational sum frequency scattering (SFS), on liquid water as well as on model lipid membranes mimicking the cell membranes.We investigate with SHS how the orientational ordering of water molecules is affected by temperature changes and nuclear quantum effects. Scattering measurements of pure water reveal that the intermolecular correlations undergo a symmetry transformation with increasing temperature. On the other hand, with increasing temperature, aqueous electrolyte solutions exhibit a diminished influence of the combined electrostatic field on the water- water correlations. This trend is predicted qualitatively by a Debye-Hückel model, but not quantitatively due to its non-inclusion of hydrogen bonding and nuclear quantum effects. These insights facilitate the elaboration of future nuclear quantum mechanical models of water.The same interactions are further studied by a comparison of pure liquid water and pure liquid carbon tetrachloride. SHS measurements and simulations reveal that transient nanoscale voids exist in water and carbon tetrachloride. The coherent SHS emissions observed in water are generated by charge density fluctuations around the cavities. These measurements strongly hint towards non-uniformity in the structure of water, on femtosecond timescale and nanometric range.Next, we turn towards the structure of water around model lipid membranes. We explore first the three-dimensional confinement of liquid water inside liposomes of various size. By analyzing SHS patterns of zwitterionic and anionic liposomes, we observe that the effect of confinement is visible on a range of a hundred nanometers, larger compared to the previously reported ranges on the order of a nanometer or about ten nanometers. The water orientational ordering at the interface as well as the surface potential of the liposomes are retrieved with a nonlinear light scattering modeling, showing that the confinement effect is mainly attributable to the hydrogen bond network. The effect is in particular 8 times larger for light water than for heavy water.Finally, we investigate how the water structure is modified around lipid monolayers and bilayers. The hydration of lipid membranes is asymmetric with respect to charge. SHS and vibrational SFS reveal that the charge hydration asymmetry is drastically different depending on the nature of the lipid layer. It depends on a delicate balance between electrostatic and hydrogen bonding interactions. The charge hydration asymmetry is further modified by the presence of charges on the lipid headgroups on both sides of the bilayers.

Soft matter probed by nonlinear scattering: self-assembly, interfaces, hydration, and long-range order

Jan Dedic

All organisms are made to a large extent of soft matter - macromolecules such as proteins and polysaccharides, or assemblies of small molecules such as lipids embedded in an aqueous environment. Understanding the role of order in soft matter presents a challenge for experimentalists because such systems are neither completely disordered nor perfectly crystalline. This is especially the case for the aqueous environment itself whose order fluctuates on femto- and pico-second time scales. In this thesis, we attempt to elucidate the presence, extent, changes, and implications of orientational order in several soft matter systems: from polyelectrolyte solutions to lipid membrane interfaces and supramolecular structures. To perform this task, we employ nonlinear optical techniques: second-harmonic and sum-frequency scattering. In the first two chapters, we explore the structure of water in aqueous polyelectrolyte solutions. Polyelectrolytes of high molecular mass are shown to induce orientational order in water that spans hundreds of nanometers. Using an adapted model of second-harmonic scattering from charged particles, we can explain the observed ordering in terms of weakly screened long-range electrostatic interactions that perturb the structure of water via charge-dipole interactions. Upon exchanging ordinary (light) water for heavy water, we observe a significant nuclear quantum effect in the induced order, indicating that bulk water-water correlations are enhanced as well by polyelectrolytes. In the following chapter, we exploit the same nuclear quantum effect in polyelectrolyte solutions to show that the polyelectrolyte-induced orientational order correlates with an anomalous increase in the reduced viscosity. We propose that the viscosity anomaly arises from enhanced order in a solvent which hinders the viscous flow. This observation represents a rare direct link between a microscopic property and a macroscopic observable. In the last two chapters, we change focus from homogeneous solutions to dispersions of liposomes which serve as models of biological membranes. In Chapter 5 we explore the binding of the protein alpha-synuclein to anionic vesicles from the perspective of interfacial water. We show that the adsorption of the protein to a vesicle decreases the order in the interfacial water by 30% but does not affect the surface nor the zeta potential. We propose that the changes in the structure of interfacial water can serve as a label-free probe of protein binding. In the last chapter, we investigate the supramolecular chemistry of inclusion complexes of methyl-Î²-cyclodextrin with various lipids. By combining multiple nonlinear scattering techniques, we show that the complexes self-assemble into micrometer-long directed fibers. Based on measurements of different lipids, we propose that the self-assembly process is driven by hydrogen bonding between the lipid headgroup and cyclodextrin. Lastly, we show that the long-range order of the self-assembled structure is transmitted into the structure of the hydrating water.

EPFL2020

Second harmonic scattering of water in a biological context

Tereza Schönfeldová

Water is one of the most abundant molecules in the universe. It forms a hydrogen bond network with unique structure and dynamics. Hydrogen bonding is highly relevant to many biological processes including membrane formation, protein folding, adsorption, and lubrication. However, the underlying molecular interactions within water and between water and membranes are difficult to study and hence not fully understood. In this thesis, we utilize second harmonic scattering (SHS) to investigate inhomogeneities in bulk water, probe structural changes in lipid bilayer membranes, and detect and characterize protein-membrane interactions. Firstly, we explore the structure of bulk water in comparison to a common model liquid, carbon tetrachloride. Combining SHS measurements and molecular dynamics simulations, we report on the nanoscale inhomogeneities in bulk water that occur on femtosecond timescales. We attribute the rise in the coherent second harmonic (SH) intensity to charge density fluctuations with enhanced nanoscale polarizabilities around transient voids. Even though the transient voids also exist in carbon tetrachloride, they do not show polarizability fluctuations and the SH response of carbon tetrachloride thus corresponds to that of a model isotropic liquid. Then, we study the structure of water around large unilamellar vesicles (LUVs), which serve as a model for cell membranes. We focus on the role of water during the lipid melting phase transition. Using SHS, differential scanning calorimetry, and temperature-dependent zeta-potential measurements, we investigate the structural changes of hydration shells around the vesicle membrane. We use lipids with phosphoserine, phosphocholine, and phosphate headgroups and observe different hydration behavior upon the lipid melting transition depending on the membrane composition. Next, we characterize the interface of membranes composed of lipids containing phosphocholine and phosphate headgroups in solutions with different CaCl2 concentrations. We observe that the SH intensity is decreasing with the addition of CaCl2, which we attribute to the formation of a condensed layer of Na+ counterions on the inner leaflet of the LUVs. Additionally, we extract the vesicle surface potential and second-order surface susceptibility from SHS data for membranes with different content of lipids with phosphate headgroups, and observe composition dependent behaviour which suggests structural membrane reorganization. We continue by exploring protein-membrane interactions using water as a contrast agent. We study a pore-forming toxin, perfringolysin O (PFO), known for selective insertion into cholesterol-rich membranes. Combining SHS measurements and cryo-electron microscopy, we compare the interaction between PFO and LUV membranes with a high and low cholesterol content. By obtaining a sub-picomolar insertion constant of PFO, we show that SHS can be used to determine protein-membrane binding constants in concentration ranges not accessible by conventional methods.Finally, we investigate the interaction of protein aerolysin and its mutants with lipid membranes. We observe changes in surface charge in the cap region of membrane-bound aerolysin at different pH of the surrounding solution. By combining SHS with single-channel measurements, we obtain the membrane binding constant of wild-type aerolysin and its mutants, and we dissect their pore-forming mechanism.

EPFL2022

Molecular insights in aqueous systems

Yixing Chen

The unique structure and dynamics of the water hydrogen (H)-bond network enable a multitude of structures and chemical reactions in both bulk solutions and at interfaces. The underlying molecular interactions between water and dissolved electrolytes, organic molecules, and nanoscale interfaces are difficult to study and hence not fully understood, especially when it involves interactions of length scales larger than one nanometer. In this thesis, we perform second order nonlinear light scattering experiments to investigate extended hydration shells (over one nanometer) and interfacial structures of buried aqueous interfaces that surround nanodroplets. We investigate the spatial range of ion-water interactions by probing the molecular structure of the water network in dilute electrolyte solutions. We find that electrolytes induce long-range, non-ion-specific orientational order in the H-bond network of water (over distances up to 70 hydration shells or 20 nm). These modifications in the water network start at electrolyte concentrations as low as 10 micromolar. The amount of induced orientational order and the concentration at which it occurs is different between light and heavy water. We link the observed ion induced changes in the bulk H-bond network of water to ion induced surface tension anomalies that occur in the same concentration range. We also study the same interactions in solutions with higher electrolyte concentrations focusing on cations. We observe specific ion effects in both the local charge distribution and water ordering in extended hydration shells of ions. Cations with larger charge densities induce larger changes in these two observables that follow a direct Hofmeister trend. The ion-induced structural changes in the water network are strongly correlated with the viscosity B-coefficient and hydration free energy of cations. These correlations facilitate constructing a better molecular model for specific ion effects in aqueous solutions. Next, we study the nanoscale hydrophobe/water interface and related interactions by probing the molecular structure of the interface formed with amphiphilic alkanols. We find that alkanols form a fluid film at the oil nanodroplet surface. Long chain alkanols show similar molecular structures and number densities at the interface. With increasing alkanol density, interfacial water loses its initial orientational alignment. The structure of interfacial oil molecules is more distorted for shorter alkanols. This interfacial structure differs significantly from those of macroscopic planar alkanol/water and alkanol/air interfaces and charged surfactant/oil/water interface, which can be explained by the balance between dispersive and H-bonding interactions. Finally, we develop a new membrane model system: 3D-lipid monolayers with tunable molecular structure on oil nanodroplets in water. This in-situ prepared system mimics the structure of lipid droplets in cells. The interfacial structure of lipids can be tuned from a tightly packed monolayer (liquid condensed phase) to a dilute one (liquid condensed/liquid expanded coexistence phase) by varying the lipid/oil/water ratio. The tunability of the chemical structure, the high surface-to-volume ratio, and the small sample volume make this system ideal for studies of molecular interactions of lipids in membranes.

EPFL2017