Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization

The dynamics and state of lipid bilayer-internal hydration water of unilamellar lipid vesicles dispersed in solutions is characterized. This study was enabled by a recently developed technique based on Overhauser dynamic nuclear polarization (DNP)-driven amplification of (1)H nuclear magnetic resonance (NMR) signal of hydration water. This technique can, in the full presence of bulk water, selectively quantify the translational dynamics of hydration water within ～10 ? around spin labels that are specifically introduced to the local volume of interest within the lipid bilayer. With this approach, the local apparent diffusion coefficients of internal water at different depths of the lipid bilayer were determined. The modulation of these values as a response to external stimuli, such as the addition of sodium chloride or ethanol and the lipid phase transitions, that alter the fluctuations of bilayer interfaces together with the activation energy values of water diffusivity shows that water is not individually and homogeneously solvating lipid's hydrocarbon tails in the lipid bilayer. We provide experimental evidence that instead, water and the lipid membrane comprise a heterogeneous system whose constituents include transient hydrophobic water pores or water structures traversing the lipid bilayer. We show how these transient pore structures, as key vehicles for passive water transport can better reconcile our experimental data with existing literature data on lipid bilayer hydration and dynamics.     

Systematic implicit solvent coarse graining of dimyristoylphosphatidylcholine lipids

We have used systematic structure‐based coarse graining to derive effective site–site potentials for a 10‐site coarse‐grained dimyristoylphosphatidylcholine (DMPC) lipid model and investigated their state point dependence. The potentials provide for the coarse‐grained model the same site–site radial distribution functions, bond and angle distributions as those computed in atomistic simulations carried out at four different lipid–water molar ratios. It was shown that there is a non‐negligible dependence of the effective potentials on the concentration at which they were generated, which is also manifested in the properties of the lipid bilayers simulated using these potentials. Thus, effective potentials computed at low lipid concentration favor to more condensed and ordered structure of the bilayer with lower average area per lipid, while potentials obtained at higher lipid concentrations provide more fluid‐like structure. The best agreement with the reference data and experiment was achieved using the set of potentials derived from atomistic simulations at 1:30 lipid:water molar ratio providing fully saturated hydration of DMPC lipids. Despite theoretical limitations of pairwise coarse‐grained potentials expressed in their state point dependence, all the resulting potentials provide a stable bilayer structure with correct partitioning of different lipid groups across the bilayer as well as acceptable values of the average lipid area, compressibility and orientational ordering. In addition to bilayer simulations, the model has proven its robustness in modeling of self‐aggregation of lipids from randomly dispersed solution to ordered bilayer structures, bicelles, and vesicles. © 2014 Wiley Periodicals, Inc.     

OPTICAL SPECTROSCOPY OF BILAYER MEMBRANES

Abstract— The absorption and fluorescence spectroscopy of natural and model bilayer lipid membranes is reviewed. Basic structural features of biological membranes and the relative advantages of black lipid membranes (BLM) and of liposomes are discussed. Theoretical considerations show that the wavelengths of absorption maxima are affected by the refractive index and dielectric constant of the medium surrounding the chromophore. Techniques of obtaining photoelectric action spectra, direct absorption spectra, and reflection spectra of BLM are described. Polarized spectra can give information about the orientation of membrane constituents and show, for example, that the porphyrin ring of chlorophyll in BLM is tilted at 45 ± 5° to the membrane surface. Absorption maxima of chlorophyll in BLM are compared with solution spectra of various chlorophyll adducts and aggregates. It is concluded that chlorophyll in BLM exists largely as solvated monomer and dimer (or oligomer), depending on concentration, and is not coordinated with water. From the theory of fluorescence spectroscopy it follows that aggregation and the polarity of the environment affect the fluorescence yield and lifetime of a membrane component, and also the wavelength of its emission maximum. The microviscosity of the membrane matrix affects the anisotropy of fluorescence. Techniques of steady-state fluorescence spectroscopy and of fluorescence lifetime measurements are reviewed. Examples of the use of fluorescent probes in membrane studies are given. Certain probes such as anilinonaphthalene sulfonate (ANS) preferentially bind to membrane proteins. The location of a probe in a particular membrane region can be pinpointed from its fluorescence yield and emission maximum. The orientation of the hydrocarbon chains of membrane lipids has been found, from fluorescence polarization of certain probes, to be normal to the membrane surface as postulated a priori on the basis of the lipid bilayer model. Anisotropy of fluorescence shows that elongated probe molecules rotate rapidly about their long axes when surrounded by phospholipids but become immobilized when bound to proteins. Changes in intensity and anisotropy of fluorescence as function of temperature have demonstrated the existence of phase transitions and phase equilibria of membrane lipids. Excimer fluorescence has been used as a measure of the available lipid core volume of membranes. Mechanisms of energy transfer between membrane components are reviewed. The theoretical dependence of energy transfer on distance and orientation for several rigid and fluid membrane models is discussed in terms of the structural information it can provide. Fluorescence sensitization resulting from energy transfer within and across bilayer membranes has been demonstrated in various systems. Quantitative measurement of energy transfer efficiency in BLM has shown that such transfer is about five times more efficient than in solutions at comparable donor-acceptor distances. Lipid membranes can be viewed as structures which maintain their components at high concentrations, in a reactive state, and at favourable orientations.     

A new method for determining the local environment and orientation of individual side chains of membrane-binding peptides

We studied here the binding of the mastoparan X peptide to a zwitterionic lipid bilayer (POPC) and demonstrated that nitrile-derivatized amino acids can be used to determine the hydration state (or change in hydration state) of specific sites of membrane-interactive peptides (upon binding). We have also shown that polarized ATR-FTIR measurements can further be used to uncover information regarding the spatial orientation of individual side chains as well as their conformational preference within the lipid bilayer.     

Characterization of the physical properties of model biomembranes at the nanometer scale with the atomic force microscope

Interaction forces and topography of mixed phospholipid-glycolipid bilayers were investigated by atomic force microscopy (AFM) in aqueous conditions with probes functionalized with self-assembled monolayers terminating in hydroxy groups. Short-range repulsive forces were measured between the hydroxy-terminated probe and the surface of the two-dimensional (2-D) solid-like domains of distearoyl-phosphatidylethanolamine (DSPE) and digalactosyldiglyceride (DGDG). The form and range of the short-range repulsive force indicated that repulsive hydration/steric forces dominate the interaction at separation distances of 0.3-1.0 nm after which the probe makes mechanical contact with the bilayers. At loads < 5 nN the bilayer was elastically deformed by the probe, while at higher loads plastic deformation of the bilayer was observed. Surprisingly, a short-range repulsive force was not observed at the surface of the 2-D liquid-like dioleoylphosphatidylethanolamine (DOPE) film, despite the identical head groups of DOPE and DSPE. This provides direct evidence for the influence of the structure and mechanical properties of lipid bilayers on their interaction forces, an effect which may be a major importance in the control of biological processes such as cell adhesion and membrane fusion. The step height measured between lipid domains in the AFM topographic images was larger than could be accounted for by the thickness and mechanical properties of the molecules. A direct correlation was observed between the repulsive force range over the lipid domains and the topographic contrast, which provides direct insight into the fundamental mechanisms of AFM imaging in aqueous solutions. This study demonstrates that chemically modified AFM probes can be used in combination with patterned lipid bilayers as a novel and powerful approach to characterize the nanometer scale chemical and physical properties of heterogeneous biosurfaces such as cell membranes.     

Alamethicin–lipid interaction studied by energy dispersive X-ray diffraction

A detailed knowledge of the interaction between bacterial membranes and antibiotics provides important information to prevent high levels of antibiotic resistance exhibited by pathogenic strains. We investigated by energy dispersive X-ray diffraction (EDXD) the structure ordering of dioleoyl-phosphatidylcholine (DOPC) lipid interacting with antimicrobial peptide alamethicin, varying the lipid/peptide (L/P) molar ratio under two different hydration levels.In conditions of full hydration (100%) we found that the bilayer thickness is constant between L/P = 20 and L/P = 80 indicating that in this range, the system has reached the threshold value for the channel formation, while at the relative hydration of 45% a linear decrease of the bilayer thickness as function of L/P was revealed. The kinetic study of the complex alamethicin–DOPC at different L/P values, shows that the Bragg peak energy variation versus the hydration time has a biexponential behavior characterized by two different time constants.     

Fluidity modulation of phospholipid bilayers by electrolyte ions: insights from fluorescence microscopy and microslit electrokinetic experiments

Fluidity and charging of supported bilayer lipid membranes (sBLMs) prepared from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) were studied by fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements at varying pH and ionic composition of the electrolyte. Measurements in neutral electrolytes (KCl, NaCl) revealed a strong correlation between the membrane fluidity and the membrane charging due to unsymmetrical water ion adsorption (OH(-) ? H(3)O(+)). The membrane fluidity significantly decreased below the isoelectric point of 3.9, suggesting a phase transition in the bilayer. The interactions of both chaotropic anions and strongly kosmotropic cations with the zwitterionic lipids were found to be related with nearly unhindered lipid mobility in the acidic pH range. While for the chaotropic anions the observed effect correlates with the increased negative net charge at low pH, no correlation was found between the changes in the membrane fluidity and charge in the presence of kosmotropic cations. We discuss the observed phenomena with respect to the interaction of the electrolyte ions with the lipid headgroup and the influence of this process on the headgroup orientation and hydration as well as on the lipid packaging.     

Ultrafast hydration dynamics in the lipidic cubic phase: Discrete water structures in nanochannels

We report here our studies of hydration dynamics of confined water in aqueous nanochannels (approximately 50 A) of the lipidic cubic phase. By systematically anchoring the hydrocarbon tails of a series of tryptophan-alkyl ester probes into the lipid bilayer, we mapped out with femtosecond resolution the profile of water motions across the nanochannel. Three distinct time scales were observed, revealing discrete channel water structures. The interfacial water at the lipid surface is well-ordered, and the relaxation dynamics occurs in approximately 100-150 ps. These dynamically rigid water molecules are crucial for global structural stability of lipid bilayers and for stabilization of anchored biomolecules in membranes. The adjacent water layers near the lipid interface are hydrogen-bonded networks and the dynamical relaxation takes 10-15 ps. This quasi-bound water motion, similar to the typical protein surface hydration relaxation, facilitates conformation flexibility for biological recognition and function. The water near the channel center is bulklike, and the dynamics is ultrafast in less than 1 ps. These water molecules freely transport biomolecules near the channel center. The corresponding orientational relaxation at these three typical locations is well correlated with the hydration dynamics and local dynamic rigidity. These results reveal unique water structures and dynamical motions in nanoconfinements, which is critical to the understanding of nanoscopic biological activities and nanomaterial properties.     

Control of the structure of self-spreading lipid membrane by changing electrolyte concentration

We have controlled the structure of self-spreading lipid bilayer membranes prepared on surface-oxidized silicon substrates by changing electrolyte concentration. Analysis of the fluorescence intensity, considering the optical interference effect, clarified the stacking structure of the lipid membrane. By varying the electrolyte concentration, we can vary the number of single multilamellar lobes adsorbed on the underlying self-spreading bilayer. This dependence of the stacking ability on the electrolyte concentration was investigated on the basis of changes in the bilayer-lobe interaction energies, including van der Waals, electrostatic double layer, and hydration interaction energies. Theoretical estimation suggests that the observed electrolyte concentration dependence can be explained by the combination of the van der Waals attractive interaction energy and the repulsive double-layer interaction energy.     

Effect of submicellar concentrations of conjugated and unconjugated bile salts on the lipid bilayer membrane

The interaction of submicellar concentrations of various physiologically important unconjugated [sodium deoxycholate (NaDC), sodium cholate (NaC)] and conjugated [sodium glycodeoxycholate (NaGDC), sodium glycocholate (NaGC), sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC)] bile salts with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles in solid gel (SG) and liquid crystalline (LC) phases was investigated using the excited-state prototropism of 1-naphthol. Steady-state and time-resolved fluorescence of the two excited-state prototropic forms of 1-naphthol indicate that submicellar bile salt concentration induces hydration of the lipid bilayer membrane into the core region. This hydration effect is a general phenomenon of the bile salts studied. The bilayer hydration efficiency of the bile salt follows the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC for both DPPC and DMPC vesicles in their SG and LC phases.     

Effect of chlorogenic acid on the phase transition in phospholipid and phospholipid/cholesterol membranes

Chlorogenic acid (CGA) is present in many plants, especially in green coffee, dry plums, and bilberries. It is an important bioactive polyphenol. Studies showed that CGA has an antioxidative, bacteriostatic, anticancer, antiviral, and anti-inflammatory activity. Despite great interest in this compound, its interaction with the lipid model membrane has not yet been investigated. To better understand the relationship between the biological activity of CGA and its interaction with biological membranes, the thermotropic behavior of model lipid membranes was investigated. The effect of CGA on the model lipid membrane, specifically on the lipid bilayer phase transitions, was examined by the combined methods: differential scanning calorimetry and fluorescence spectroscopy. In particular, the degree of packing order of the hydrophilic phase of the lipid bilayer was determined using the fluorimetric method with Laurdan and Prodan probes, while the fluorescence anisotropy of the hydrophobic phase with the DPH and TMA-DPH probes. The results of the study show that CGA incorporates mainly into the hydrophilic part of membrane, changing the packing order of the polar heads of lipids. No significant changes were recorded in membrane fluidity of the hydrophobic membrane region, for the fluorescence anisotropy practically did not change. One can thus infer that CGA does not penetrate deep into the hydrophobic area of the membrane.     

Hydrolysis of fluid supported membrane islands by phospholipase A(2): Time-lapse imaging and kinetic analysis

The activity of phospholipase A(2) (PLA(2)) which catalyzes the hydrolysis of phospholipids into free fatty acids and lysolipids, depends on the structure and thermodynamic state of the membrane. To further understand how the substrate conformation correlates with enzyme activity, model systems that are based on time-resolved membrane microscopy are needed. We demonstrate a methodology for preparing and investigating the dynamics of fluid supported phospholipid membranes hydrolyzed by snake venom PLA(2). The method uses quantitative analysis of time-lapse fluorescence images recording the evolution of fluid bilayer islands during hydrolysis. In order to minimize interactions with the support surface, we use double bilayer islands situated on top of a complete primary supported membrane prepared by hydration of spincoated lipid films. Our minimal kinetic analysis describes adsorption of enzyme to the membrane in terms of the Langmuir isotherm as well as enzyme kinetics. We use two related models assuming hydrolysis to occur either at the perimeter or at the surface of the membrane island. We find that the adsorption constant is similar for the two cases, while the estimated turnover rate is markedly different. The PLA(2) concentration series is measured in the absence and presence of beta-cyclodextrin which forms water soluble complexes with the reaction products. The results demonstrate the versatility of double bilayer islands as a membrane model system and introduces a new method for quantifying the kinetics of lipase activity on membranes by directly monitoring the evolution in substrate morphology.     

Heating and temperature gradients of lipid bilayer samples induced by RF irradiation in MAS solid‐state NMR experiments

The MAS solid‐state NMR has been a powerful technique for studying membrane proteins within the native‐like lipid bilayer environment. In general, RF irradiation in MAS NMR experiments can heat and potentially destroy expensive membrane protein samples. However, under practical MAS NMR experimental conditions, detailed characterization of RF heating effect of lipid bilayer samples is still lacking. Herein, using ¹H chemical shift of water for temperature calibration, we systematically study the dependence of RF heating on hydration levels and salt concentrations of three lipids in MAS NMR experiments. Under practical ¹H decoupling conditions used in biological MAS NMR experiments, three lipids show different dependence of RF heating on hydration levels as well as salt concentrations, which are closely associated with the properties of lipids. The maximum temperature elevation of about 10 °C is similar for the three lipids containing 200% hydration, which is much lower than that in static solid‐state NMR experiments. The RF heating due to salt is observed to be less than that due to hydration, with a maximum temperature elevation of less than 4 °C in the hydrated samples containing 120 mmol l^?1 of salt. Upon RF irradiation, the temperature gradient across the sample is observed to be greatly increased up to 20 °C, as demonstrated by the remarkable broadening of ¹H signal of water. Based on detailed characterization of RF heating effect, we demonstrate that RF heating and temperature gradient can be significantly reduced by decreasing the hydration levels of lipid bilayer samples from 200% to 30%. Copyright © 2016 John Wiley & Sons, Ltd.     

Photolabile and paramagnetic reagents for the investigation of transmembrane signaling events.

To investigate the dynamics of membrane processes that may be integral components of specific transmembrane signaling events we have synthesized several novel paramagnetic probes and their photoreactive counterparts. The structure of these probes was designed to (1) restrict "flipping" across the membrane bilayer; (2) contain paramagnetic or photoreactive moieties that could be placed at specific depths within the bilayer; (3) provide information about membrane structure as well as dynamics of protein movement; and (4) in the case of the photoreactive probes, be of high specific radioactivity. The molecules described in this paper consist of amino acid, dipeptide, or carbohydrate groups attached to arylazide- or nitroxide-bearing fatty acids. The synthesis and initial characterization of these membrane probes is described.     

Retinoid chromophores as probes of membrane lipid order

There is a great need for development of independent methods to study the structure and function of membrane-associated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.     

Sensitivity of hydrogen bond lifetime dynamics to the presence of ethanol at the interface of a phospholipid bilayer

Atomistic molecular dynamics simulations of a fully hydrated liquid crystalline lamellar phase of a dimyrystoylphosphatidylcholine lipid bilayer containing ethanol at 1:1 composition as well as of the pure lamellar phase of the bilayer have been performed. Detailed analyses have been carried out to investigate the effects of ethanol, if any, on the lifetime dynamics of lipid-water and water-water hydrogen bonds in the hydration layer of the lipid headgroups. The nonexponential hydrogen bond lifetime correlation functions have been analyzed by using the formalism of Luzar and Chandler, which allowed the identification of the bound states at the bilayer interface and the quantification of the dynamic equilibrium between the bound and the free water molecules, in terms of time-dependent relaxation rates. The calculations show that the overall relaxation of phosphate-water hydrogen bonds is faster in the presence of ethanol. Studies of the residence time and the number fluctuation of the hydration layer water molecules reveal that the presence of ethanol molecules decreases the rigidity of the lipid hydration layer.     

Microchannel device using self-spreading lipid bilayer as molecule carrier

We propose a microchannel device that employs a surface-supported self-spreading lipid bilayer membrane as a molecule carrying medium. The device has a micropattern structure fabricated on a SiO2 surface by photolithography, into which a self-spreading lipid bilayer membrane is introduced as the carrier medium. This system corresponds to a microchannel with a single lipid bilayer membrane height of approximately 5 nm, compared with conventional micro-fluidic channels that have a section height and width of at least several microm. The device is beneficial for detecting intermolecular interactions when molecules carried by the self-spreading lipid bilayer collide with each other in the microchannel. The validity of the device was confirmed by observing the fluorescence resonance energy transfer (FRET) between two dye molecules, coumarin and fluorescein.     

Role of nanomechanical properties in the tribological performance of phospholipid biomimetic surfaces

The role of phospholipid bilayers in controlling and reducing frictional forces between biological surfaces is investigated by three complementary experiments: friction forces are measured using a homemade tribometer, mechanical resistance to indentation is measured by AFM, and lipid bilayer degradation is controlled in situ during friction testing using fluorescence microscopy. DPPC lipid bilayers in the solid phase generate friction coefficients as low as 0.002 (comparable to that found for cartilage) that are stable through time. DOPC bilayers formed by the vesicle fusion method or the adsorption of mixed micelles generate higher friction coefficients. These coefficients increased through time, during which the bilayers degraded. The friction coefficient is correlated with the force needed to penetrate the bilayer with the AFM tip. With only one bilayer in the contact region, the friction increased to a similar value of about 0.08 for the DPPC and DOPC. Our study therefore shows that good mechanical stability of the bilayers is essential and suggests that the low friction coefficient is ensured by the hydration layers between adjacent lipid bilayers.     

Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers

The lipid membranes found in archaea have high bilayer stability and low permeability. The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains. In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively. We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers. We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC. The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data.