Comparative Study of Latanoprost Drug Delivery Systems for Glaucoma Treatment and Their Interaction with the Tear Film Lipid Layer Models.

This study investigates the interaction of two approved and one newly developed latanoprost formulation with in vitro and in silico models of the tear film and tear film lipid layer (TFLL). Latanoprost, a prostaglandin analogue used for intraocular elevated pressure treatment, is topically delivered by nanocarriers within aqueous solutions or emulsions. The study focuses on the impact of these carriers on drug interactions with the tear film and their effect on the TFLL. Three different types of latanoprost carriers, micellar, nanoemulsion, and polymer-based, were compared, and each revealed distinct interaction patterns with the TFLL. Surface pressure kinetics demonstrated a rapid increase for the benzalkonium chloride formulation and a slow rise for the preservative-free variants. Visualization of the acellular in vitro TFLL model revealed different patterns of incorporation for each formulation, indicating unique interaction mechanisms. Molecular dynamics simulations further revealed different mechanisms of drug release in the TFLL between micellar and nanoemulsion formulations. In-depth examination highlighted the role of triglyceride molecules in replenishing the nonpolar layer of the TFLL, which suggests potential improvements in ocular surface compatibility by adjusting the quality and concentration of the oily phase. These findings suggest the potential for optimizing latanoprost formulations by tuning the oily phase-to-surfactant ratio and selecting suitable surfactants.

Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed nonequilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multicomponent lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

Properties of Lipid Models of Lung Surfactant Containing Cholesterol and Oxidized Lipids: A Mixed Experimental and Computational Study.

We introduce and study a multicomponent lipid film mimicking lipid composition of the human lung surfactant. It consists of phospholipids with various lipid headgroups and tail saturation. Furthermore, it includes cholesterol and oxidized lipids. Langmuir trough and fluorescence microscopy experiments are combined with fully atomistic molecular dynamics simulations. The considered lipid mixtures form complex interfacial films with properties modulated by lateral compression. Cholesterol laterally condenses, and oxidized lipids laterally expand the films; both types of molecules increase film miscibility. Oxidized lipids also alter the lipid-water interface enhancing film hydration; this effect can be partially reversed by cholesterol. Regarding presentation of different chemical moieties toward the aqueous subphase, the zwitterionic phosphatidylcholine groups dominate at the lipid-water interface, while both the negatively charged phosphatidylglycerol and hydroxyl group of cholesterol are less exposed. The investigated synthetic lipid-only mimic of the lung surfactant may serve as a basis for further studies involving nonlipid pulmonary surfactant components.

Tail-Oxidized Cholesterol Enhances Membrane Permeability for Small Solutes.

Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7ß-hydroxycholesterol (7ß-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane-bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7ß-OH-chol which represents ring-oxidized sterols.

Improving Stability of Tear Film Lipid Layer via Concerted Action of Two Drug Molecules: A Biophysical View.

The tear film at the ocular surface is covered by a thin layer of lipids. This oily phase stabilizes the film by decreasing its surface tension and improving its viscoelastic properties. Clinically, destabilization and rupture of the tear film are related to dry eye disease and are accompanied by changes in the quality and quantity of tear film lipids. In dry eye, eye drops containing oil-in-water emulsions are used for the supplementation of lipids and surface-active components to the tear film. We explore in detail the biophysical aspects of interactions of specific surface-active compounds, cetalkonium chloride and poloxamer 188, which are present in oil-in-water emulsions, with tear lipids. The aim is to better understand the macroscopically observed eye drops-tear film interactions by rationalizing them at the molecular level. To this end, we employ a multi-scale approach combining experiments on human meibomian lipid extracts, measurements using synthetic lipid films, and in silico molecular dynamics simulations. By combining these methods, we demonstrate that the studied compounds specifically interact with the tear lipid film enhancing its structure, surfactant properties, and elasticity. The observed effects are cooperative and can be further modulated by material packing at the tear-air interface.

Mixed DPPC/POPC Monolayers: All-atom Molecular Dynamics Simulations and Langmuir Monolayer Experiments.

To elucidate the consequences of the saturated-unsaturated nature of lipid surface films, monolayers formed by an equimolar mixture of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipids are investigated in a wide range of surface pressures. As such mixtures share some features with naturally-occurring surfactants, for example the lung surfactant, the systems are studied at the temperature relevant for human body. All-atom molecular dynamics simulations and Langmuir trough experiments are employed. The binary lipid mixture is compared with the corresponding one-component systems. Atomistic-level alterations of monolayer molecular properties upon lateral compression are scrutinized. These involve elevation of lateral ordering of lipid chains, modulation of chain and headgroup orientation, and reduction of lipid hydration. The presence of the unsaturated POPC in the DPPC/POPC mixture reduces the liquid expanded-liquid condensed coexistence region and moderates the phase transition. Simulations predict that nanoscale lipid de-mixing occurs with small transient DPPC clusters emerging due to local fluctuations of the lateral lipid arrangement. A vertical sorting of lipids induced by lateral compression is also observed, with DPPC transferred toward the water phase. Both the conformational lipid alterations due to monolayer compression as well as the existence of lateral dynamic inhomogeneities of the lipid film are potentially pertain to dynamic and non-homogeneous lipid interfacial systems.

Phospholipid lateral diffusion in phosphatidylcholine-sphingomyelin-cholesterol monolayers; effects of oxidatively truncated phosphatidylcholines.

Oxidative stress is involved in a number of pathological conditions and the generated oxidatively modified lipids influence membrane properties and functions, including lipid-protein interactions and cellular signaling. Brewster angle microscopy demonstrated oxidatively truncated phosphatidylcholines to promote phase separation in monolayers of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). More specifically, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), was found to increase the miscibility transition pressure of the SM/Chol-phase. Lateral diffusion of lipids is influenced by a variety of membrane properties, thus making it a sensitive parameter to observe the coexistence of different lipid phases, for instance. The dependence on lipid lateral packing of the lateral diffusion of fluorophore-containing phospholipid analogs was investigated in Langmuir monolayers composed of POPC, SM, and Chol and additionally containing oxidatively truncated phosphatidylcholines, using fluorescence correlation spectroscopy (FCS). To our knowledge, these are the first FCS results on miscibility transition in ternary lipid monolayers, confirming previous results obtained using Brewster angle microscopy on such lipid monolayers. Wide-field fluorescence microscopy was additionally employed to verify the transition, i.e. the loss and reformation of SM/Chol domains.

Experimental determination and computational interpretation of biophysical properties of lipid bilayers enriched by cholesteryl hemisuccinate.

Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water-membrane interface. The implications of these findings for considerations of protein-cholesterol interactions are briefly discussed.

Comprehensive portrait of cholesterol containing oxidized membrane.

Biological membranes are under significant oxidative stress caused by reactive oxygen species mostly originating during cellular respiration. Double bonds of the unsaturated lipids are most prone to oxidation, which might lead to shortening of the oxidized chain and inserting of terminal either aldehyde or carboxylic group. Structural rearrangement of oxidized lipids, addressed already, is mainly associated with looping back of the hydrophilic terminal group. This contribution utilizing dual-focus fluorescence correlation spectroscopy and electron paramagnetic resonance as well as atomistic molecular dynamics simulations focuses on the overall changes of the membrane structural and dynamical properties once it becomes oxidized. Particularly, attention is paid to cholesterol rearrangement in the oxidized membrane revealing its preferable interaction with carbonyls of the oxidized chains. In this view cholesterol seems to have a tendency to repair, rather than condense, the bilayer.

Peripheral and integral membrane binding of peptides characterized by time-dependent fluorescence shifts: focus on antimicrobial peptide LAH4.

Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

Pairing of cholesterol with oxidized phospholipid species in lipid bilayers.

We claim that (1) cholesterol protects bilayers from disruption caused by lipid oxidation by sequestering conical shaped oxidized lipid species such as 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PZPC) away from phospholipid, because cholesterol and the oxidized lipid have complementary shapes and (2) mixtures of cholesterol and oxidized lipids can self-assemble into bilayers much like lysolipid­cholesterol mixtures. The evidence for bilayer protection comes from molecular dynamics (MD) simulations and dynamic light scattering (DLS) measurements. Unimodal size distributions of extruded vesicles (LUVETs) made up of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and PZPC containing high amounts of PZPC are only obtained when cholesterol is present in high concentrations. In simulations, bilayers containing high amounts of PZPC become porous, unless cholesterol is also present. The protective effect of cholesterol on oxidized lipids has been observed previously using electron paramagnetic resonance (EPR) and electron microscopy imaging of vesicles. The evidence for the pairing of cholesterol and PZPC comes mainly from correlated 2-D density and thickness plots from simulations, which show that these two molecules co-localize in bilayers. Further evidence that the two molecules can cohabitate comes from self-assembly simulations, where we show that cholesterol-oxidized lipid mixtures can form lamellar phases at specific concentrations, reminiscent of lysolipid­cholesterol mixtures. The additivity of the packing parameters of cholesterol and PZPC explains their cohabitation in a planar bilayer. Oxidized lipids are ubiquitously present in significant amounts in high- and low-density lipoprotein (HDL and LDL) particles, diseased tissues, and in model phospholipid mixtures containing polyunsaturated lipids. Therefore, our hypothesis has important consequences for cellular cholesterol trafficking; diseases related to oxidized lipids, and to biophysical studies of phase behaviour of cholesterol-containing phospholipid mixtures.

Biophysics of lipid bilayers containing oxidatively modified phospholipids: insights from fluorescence and EPR experiments and from MD simulations.

This review focuses on the influence of oxidized phosphatidylcholines (oxPCs) on the biophysical properties of model membranes and is limited to fluorescence, EPR, and MD studies. OxPCs are divided into two classes: A) hydroxy- or hydroperoxy-dieonyl phospatidylcholines, B) phospatidylcholines with oxidized and truncated chains with either aldehyde or carboxylic group. It was shown that the presence of the investigated oxPCs in phospholipid model membranes may have the following consequences: 1) decrease of the lipid order, 2) lowering of phase transition temperatures, 3) lateral expansion and thinning of the bilayer, 4) alterations of bilayer hydration profiles, 5) increased lipid mobility, 6) augmented flip-flop, 7) influence on the lateral phase organisation, and 8) promotion of water defects and, under extreme conditions (i.e. high concentrations of class B oxPCs), disintegration of the bilayer. The effects of class A oxPCs appear to be more moderate than those observed or predicted for class B. Many of the abovementioned findings are related to the ability of the oxidized chains of certain oxPCs to reorient toward the water phase. Some of the effects appear to be moderated by the presence of cholesterol. Although those biophysical alternations are found at oxPC concentrations higher than the total oxPC concentrations found under physiological conditions, certain organelles may reach such elevated oxPC concentrations locally. It is a challenge for the future to correlate the biophysics of oxidized phospholipids to metabolic studies in order to define the significance of the findings presented herein for pathophysiology. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

Molecular rheometry: direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

Stealthy Player in Lipid Experiments? EDTA Binding to Phosphatidylcholine Membranes Probed by Simulations and Monolayer Experiments.

Ethylenediaminetetraacetic acid (EDTA) is frequently used in lipid experiments to remove redundant ions, such as Ca2+, from the sample solution. In this work, combining molecular dynamics (MD) simulations and Langmuir monolayer experiments, we show that on top of the expected Ca2+ depletion, EDTA anions themselves bind to phosphatidylcholine (PC) monolayers. This binding, originating from EDTA interaction with choline groups of PC lipids, leads to the adsorption of EDTA anions at the monolayer surface and concentration-dependent changes in surface pressure as measured by monolayer experiments and explained by MD simulations. This surprising observation emphasizes that lipid experiments carried out using EDTA-containing solutions, especially of high concentrations, must be interpreted very carefully due to potential interfering interactions of EDTA with lipids and other biomolecules involved in the experiment, e.g., cationic peptides, that may alter membrane-binding affinities of studied compounds.

Latanoprost incorporates in the tear film lipid layer: An experimental and computational model study.

Glaucoma is a leading cause of blindness worldwide, with elevated intraocular pressure being a major risk factor for its development and progression. First-line treatment for glaucoma relies on the administration of prostaglandin analogs, with latanoprost being the most widely used. However, before latanoprost reaches the cornea, it must pass through the tear film and tear film lipid layer (TFLL) on the ocular surface. Given the significant lipophilicity of latanoprost, we hypothesize that TFLL could, to a certain extent, act as a reservoir for latanoprost, releasing it on longer time scales, apart from the fraction being directly delivered to the cornea in a post-instillation mechanism. We investigated this possibility by studying latanoprost behavior in acellular in vitro TFLL models. Furthermore, we employed in silico molecular dynamics simulations to rationalize the experimental results and obtain molecular-level insight into the latanoprost-TFLL interactions. Our experiments demonstrated that latanoprost indeed accumulates in the TFLL models, and our simulations explain the basis of the accumulation mechanism. These results support the hypothesis that TFLL can serve as a reservoir for latanoprost, facilitating its prolonged release. This finding could have significant implications for optimizing glaucoma treatment, especially in the development of new drug delivery systems targeting the TFLL.

Influence of BAKs on tear film lipid layer: In vitro and in silico models.

Benzalkonium chloride (BAK) compounds are commonly used in topical ophthalmic products as preservatives and stabilizers. BAK mixtures containing several compounds with different alkyl chain lengths are typically used. However, in chronic eye conditions, such as dry eye disease and glaucoma, the accumulation of adverse effects of BAKs was observed. Hence, preservative-free eye drops formulations are preferred. On the other hand, selected long-chain BAKs, particularly cetalkonium chloride, exhibit therapeutic functions, promoting epithelium wound healing and tear film stability. Nevertheless, the mechanism of BAKs influence on the tear film is not fully understood. By employing in vitro experimental and in silico simulation techniques, we elucidate the action of BAKs and demonstrate that long-chain BAKs accumulate in the lipid layer of the tear film model, stabilizing it in a concentration-dependent fashion. In contrast, short-chain BAKs interacting with the lipid layer compromise the tear film model stability. These findings are relevant for topical ophthalmic drug formulation and delivery in the context of selecting proper BAK species and understanding the dose dependency for tear film stability.

Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures.

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

Interactions of polar lipids with cholesteryl ester multilayers elucidate tear film lipid layer structure.

PURPOSE: The tear film lipid layer (TFLL) covers the tear film, stabilizing it and providing a protective barrier against the environment. The TFLL is divided into polar and non-polar sublayers, but the interplay between lipid classes in these sublayers and the structure-function relationship of the TFLL remains poorly characterized. This study aims to provide insight into TFLL function by elucidating the interactions between polar and non-polar TFLL lipids at the molecular level. METHODS: Mixed films of polar O-acyl-ω-hydroxy fatty acids (OAHFA) or phospholipids and non-polar cholesteryl esters (CE) were used as a model of the TFLL. The organization of the films was studied by using a combination of Brewster angle and fluorescence microscopy in a Langmuir trough system. In addition, the evaporation resistance of the lipid films was evaluated. RESULTS: Phospholipids and OAHFAs induced the formation of a stable multilamellar CE film. The formation of this film was driven by the interdigitation of acyl chains between the monolayer of polar lipids and the CE multilayer lamellae. Surprisingly, the multilayer structure was destabilized by both low and high concentrations of polar lipids. In addition, the CE multilayer was no more effective in resisting the evaporation of water than a polar lipid monolayer. CONCLUSIONS: Formation of multilamellar films by major tear film lipids suggest that the TFLL may have a similar structure. Moreover, in contrast to the current understanding, polar TFLL lipids may not mainly act by stabilizing the non-polar TFLL sublayer, but through a direct evaporation resistant effect.

Mixed polar-nonpolar lipid films as minimalistic models of Tear Film Lipid Layer: A Langmuir trough and fluorescence microscopy study.

The Tear Film Lipid Layer (TFLL) covering the surface of the aqueous film at human cornea forms a first barrier between the eye and environment. Its alterations are related to dry eye disease. TFLL is formed by a complex mixture of lipids, with an excess of nonpolar components and a minor fraction of polar molecules. Its thickness is up to 160 nm, hence a multilayer-like structure of TFLL is assumed. However, details of TFLL organization are mostly unavailable in vivo due to the dynamic nature of the human tear film. To overcome this issue, we employ a minimalistic in vitro lipid model of TFLL. We study its biophysical characteristics by using a combination of the Langmuir trough with fluorescence microscopy. The model consists of two-component polar-nonpolar lipid films with a varying component ratio spread on the aqueous subphase at physiologically relevant temperature. We demonstrate that the model lipid mixture undergoes substantial structural reorganization as a function of lateral pressure and polar to nonpolar lipid ratio. In particular, the film is one-molecule-thick and homogenous under low lateral pressure. Upon compression, it transforms into a multilayer structure with inhomogeneities in the form of polar-nonpolar lipid assemblies. Based on this model, we hypothesize that TFLL in vivo has a duplex polar-nonpolar structure and it contains numerous mixed lipid aggregates formed because of film restructuring. These findings, despite the simplified character of the model, seem relevant for TFLL physiology as well as for understanding pathological conditions related to the lipids of the tear film.

Nanosecond time-dependent Stokes shift at the tunnel mouth of haloalkane dehalogenases.

The tunnel mouths are evolutionally the most variable regions in the structures of haloalkane dehalogenases originating from different bacterial species, suggesting their importance for adaptation of enzymes to various substrates. We decided to monitor the dynamics of this particular region by means of time-resolved fluorescence spectroscopy and molecular dynamic simulations. To label the enzyme specifically, we adapted a novel procedure that utilizes a coumarin dye containing a halide-hydrocarbon linker, which serves as a substrate for enzymatic reaction. The procedure leads to a coumarin dye covalently attached and specifically located in the tunnel mouth of the enzyme. In this manner, we stained two haloalkane dehalogenase mutants, DbjA-H280F and DhaA-H272F. The measurements of time-resolved fluorescence anisotropy, acrylamide quenching, and time-resolved emission spectra reveal differences in the polarity, accessibility and mobility of the dye and its microenvironment for both of the mutants. The obtained experimental data are consistent with the results obtained by molecular dynamics calculations and correlate with the anatomy of the tunnel mouths, which were proposed to have a strong impact on the catalytic activity and specificity of the examined mutants. Interestingly, the kinetics of the recorded time-dependent Stokes shift is unusual slow; it occurs on the nanosecond time-scale, suggesting that the protein dynamics is extremely slowed down at the region involved in the exchange of ligands between the active-site cavity and bulk solvent.