Direct submolecular scale imaging of mesoscale molecular order in supported dipalmitoylphosphatidylcholine bilayers

Supported dipalmitoylphosphatidylcholine (DPPC) bilayers are widely used membrane systems in biophysical and biochemical studies. Previously, short-range positional and orientational order of lipid headgroups of supported DPPC bilayers was observed at room temperature using low deflection noise frequency modulation atomic force microscopy (FM-AFM). While this ordering was supported by X-ray diffraction studies, it conflicted with diffusion coefficient measurements of gel-phase bilayers determined from fluorescence photobleaching experiments. In this work, we have directly imaged mica-supported DPPC bilayers with submolecular resolution over scan ranges up to 146 nm using low deflection noise FM-AFM. Both orientational and positional molecular ordering were observed in the mesoscale, indicative of crystalline order. We discuss these results in relation to previous biophysical studies and propose that the mica support induces mesoscopic crystalline order of the DPPC bilayer at room temperature. This study also demonstrates the recent advance in the scan range of submolecular scale AFM imaging.     

Free pyrene probes in gel and fluid membranes: perspective through atomistic simulations

We consider the properties of free pyrene probes inside gel- and fluidlike phospholipid membranes and unravel their influence on membrane properties. For this purpose, we employ atomic-scale molecular dynamics simulations at several temperatures for varying pyrene concentrations. Molecular dynamics simulations show that free pyrene molecules prefer to be located in the hydrophobic acyl chain region close to the glycerol group of lipid molecules. Their orientation is shown to depend on the phase of the membrane. In the fluid phase, pyrenes favor orientations where they are standing upright in parallel to the membrane normal, while, in the gel phase, the orientation is affected by the tilt of lipid acyl chains. Pyrenes are found to locally perturb membrane structure, while the nature of perturbations in the gel and fluid phases is completely different. In the gel phase, pyrenes break the local packing of lipids and decrease the ordering of lipid acyl chains around them, while, in the fluid phase, pyrenes increase the ordering of nearby acyl chains, thus having an opposite effect. Interestingly, this proposes a similarity to effects induced by cholesterol on structural membrane properties above and below the gel-fluid transition temperature. Further studies express a view that the orientational ordering of pyrene is not a particularly good measure of the acyl chain ordering of lipids. While pyrene ordering provides the correct qualitative behavior of acyl chain ordering in the fluid phase, its capability to predict the correct temperature dependence is limited.     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.     

Interaction of tetraphenyl-porphyrin derivatives with DPPC-liposomes: an EPR study

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC. The effect on membrane fluidity proved to be stronger with the asymmetrical porphyrin derivative than with the symmetrical one. The rigidity increased when the spin label was near lipid head groups. The difference observed between control and porphyrin-treated samples when measured below the main lipid transition temperature disappeared at higher temperature. When the spin label was near the end of the hydrophobic tails, the symmetrical porphyrin derivative caused increase in fluidity, while the asymmetrical one slightly decreased it. To explain this phenomenon we propose that the asymmetrical derivative exerts a stronger ordering effect caused by its fluorophenyl group located at the level of the lipid heads, which is attenuated to the hydrophobic tails. The perturbing effect of the symmetric derivative could not lead to similar extent of ordering at the head groups and looses the hydrocarbon chains deeper in the membrane.     

Dipolar ordering in the ripple phases of molecular-scale models of lipid membranes

Symmetric and asymmetric ripple phases have been observed to form in molecular dynamics simulations of a simple molecular-scale lipid model. The lipid model consists of an dipolar head group and an ellipsoidal tail. Within the limits of this model, an explanation for generalized membrane curvature is a simple mismatch in the size of the heads with the width of the molecular bodies. The persistence of a bilayer structure requires strong attractive forces between the head groups. One feature of this model is that an energetically favorable orientational ordering of the dipoles can be achieved by out-of-plane membrane corrugation. The corrugation of the surface stabilizes the long range orientational ordering for the dipoles in the head groups which then adopt a bulk anti-ferroelectric state. We observe a common feature of the corrugated dipolar membranes: the wave vectors for the surface ripples are always found to be perpendicular to the dipole director axis.     

Molecular aspects of the interaction between plants sterols and DPPC bilayers: an experimental and theoretical approach

The effect of sterols composition in a lipid bilayer was investigated on membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mixtures with the plant sterols β-sitosterol and stigmasterol. Differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization and infrared spectroscopy studies showed that both sterols changed the packing of the membrane and the hydration of the polar headgroup of the phospholipids, disordering the gel phase and, vice versa, ordering the membrane in the liquid crystalline phase. In all cases some differences among β-sitosterol and stigmasterol could be observed, being β-sitosterol slightly more efficient than stigmasterol in ordering a fluid membrane, bringing the membrane to a more packed liquid ordered phase. Molecular dynamic simulations were carried out to better characterize the distinct behavior of both sterols in a DPPC-membrane. The calculated parameters agreed quite well with the experimental results and a molecular model is proposed to explain differences in the sterols molecules and their effect on the DPPC-bilayer.     

Curvature-modulated phase separation in lipid bilayer membranes

Cellular membranes exhibit a variety of controlled curvatures, with filopodia, microvilli, and mitotic cleavage furrows being only a few of many examples. Coupling between local curvature and chemical composition in membranes could provide a means of mechanically controlling the spatial organization of membrane components. Although this concept has surfaced repeatedly over the years, controlled experimental investigations have proven elusive. Here, we introduce an experimental platform, in which microfabricated surfaces impose specific curvature patterns onto lipid bilayers, that allows quantification of mechanochemical couplings in membranes. We find that, beyond a critical curvature value, membrane geometry governs the spatial ordering of phase-separated domain structures in membranes composed of cholesterol and phospholipids. The curvature-controlled ordering, a consequence of the distinct mechanical properties of the lipid phases, makes possible a determination of the bending rigidity difference between cholesterol-rich and cholesterol-poor lipid domains. These observations point to a strong coupling between mechanical bending and chemical organization that should have wide-reaching consequences for biological membranes. Curvature-mediated patterning may also be useful in controlling complex fluids other than biomembranes.     

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM₁-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca²⁺ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.     

Small-scale structure in fluid cholesterol–lipid bilayers

Cholesterol is the single most abundant molecule in animal plasma membranes, in the range of 20–30 mol%, where it is known to modulate the lipid-bilayer component of the membrane and lead to increased mechanical stability, lower permeability, larger thickness, and a distinct lateral organization. The phase equilibria of membranes with cholesterol and the associated large- and small-scale structure have turned out to be a particularly elusive problem. With the proposal that lipid domains and so-called ‘rafts’, characterized by high local levels of cholesterol in a liquid-ordered phase, are important for a wide range of cellular functions, an understanding and a quantitative assessment of the nature of these cholesterol-induced structures and their types of ordering have become urgent. Recent progress in neutron diffraction studies of lipid–cholesterol model membranes has now revealed details of the lateral ordering, and combined with earlier molecular model studies a picture emerges of the membrane as a locally structured liquid with small ordered ‘domains’ of a highly dynamic nature.     

Free Energy of Bare and Capped Gold Nanoparticles Permeating through a Lipid Bilayer

Herein, we study the permeation free energy of bare and octane‐thiol‐capped gold nanoparticles (AuNPs) translocating through a lipid membrane. To investigate this, we have pulled the bare and capped AuNPs from bulk water to the membrane interior and estimated the free energy cost. The adsorption of the bare AuNP on the bilayer surface is energetically favorable but further loading inside it requires energy. However, the estimated free‐energy barrier for loading the capped AuNP into the lipid membrane is much higher compared to bare AuNP. We also demonstrate the details of the permeation process of bare and capped AuNPs. Bare AuNP induces the curvature in the lipid membrane whereas capped AuNP creates an opening in the interacting monolayer and get inserted into the membrane. The insertion of capped AuNP induces a partial unzipping of the lipid bilayer, which results in the ordering of the local lipids interacting with the nanoparticle. However, bare AuNP disrupts the lipid membrane by pushing the lipid molecules inside the membrane. We also analyze pore formation due to the insertion of capped AuNP into the membrane, which results in water molecules penetrating the hydrophobic region.     

Manipulating energy landscapes to tune ordering in biotemplated nanoparticle arrays

Two-dimensional non-close-packed crystals of the protein streptavidin, grown on phospholipid membranes, can serve as nanoscale templates capable of directing the formation of ordered nanoparticle arrays through site-specific electrostatic adsorption. Here we examine the effects of both interparticle and nanoparticle/lipid membrane electrostatic interactions on the degree of structural order exhibited by the templated nanoparticle array. Interparticle electrostatic repulsion is shown to have only marginal influence on nanoparticle ordering. In contrast, the degree of order exhibited by the templated array can be tuned by controlling the charge on the lipid membrane. Analysis of the local and global structure of arrays generated with negatively charged gold nanoparticles (～6 nm) indicate improved long-range order when the lipid membrane supporting the protein crystal is derived from cationic lipid molecules as opposed to zwitterionic phospholipids. Furthermore, as nanoparticle size is reduced (～3 nm), the presence of a charged lipid membrane is found to be essential, as smaller particles do not adhere to streptavidin crystals grown on zwitterionic membranes. These findings demonstrate that the composition of the lipid support can influence the efficacy of directed-assembly processes which utilize protein templates and are important results toward enhancing control over bottom-up nanofabrication applications.     

Unraveling dendrimer translocation across cell membrane mimics

Poly(amidoamine) (PAMAM) dendrimers are promising candidates in several applications within the medical field. However, it is still to date not fully understood whether they are able to passively translocate across lipid bilayers. Recently, we used fluorescence microscopy to show that PAMAM dendrimers induced changes in the permeability of lipid membranes but the dendrimers themselves could not translocate to be released into the vesicle lumen. Because of the lack of resolution, these experiments could not assess whether the dendrimers were able to translocate but remained attached to the membrane. Using quartz crystal microbalance with dissipation monitoring and neutron reflectivity, a structural investigation was performed to determine how dendrimers interact with zwitterionic and negatively charged lipid bilayers. We hereby show that dendrimers adsorb on top of lipid bilayers without significant dendrimer translocation, regardless of the lipid membrane surface charge. Thus, most likely dendrimers are actively transported through cell membranes by protein-mediated endocytosis in agreement with previous cell studies. Finally, the higher activity of PAMAM dendrimers for phosphoglycerol-containing membranes is in line with their high antimicrobial activity against Gram-negative bacteria.     

Peptide meets membrane: Investigating peptide-lipid interactions using small-angle scattering techniques

Peptide–lipid interactions play an important role in defining the mode of action of drugs and the molecular mechanism associated with many diseases. Model membranes consisting of simple lipid mixtures mimicking real cell membranes can provide insight into the structural and dynamic aspects associated with these interactions. Small-angle scattering techniques based on X-rays and neutrons (SAXS/SANS) allow in situ determination of peptide partition and structural changes in lipid bilayers in vesicles with relatively high resolution between 1-100 nm. With advanced instrumentation, time-resolved SANS/SAXS can be used to track equilibrium and nonequilibrium processes such as lipid transport and morphological transitions to time scales down to a millisecond. In this review, we provide an overview of recent advances in the understanding of complex peptide–lipid membrane interactions using SAXS/SANS methods and model lipid membrane unilamellar vesicles. Particular attention will be given to the data analysis, possible pitfalls, and how to extract quantitative information using these techniques.     

Effect of double bond position on lipid bilayer properties: insight through atomistic simulations

Phosphatidylcholines (PCs) are among the most common phospholipids in plasma membranes. Their structural and dynamic properties are known to be strongly affected by unsaturation of lipid hydrocarbon chains, yet the role of the exact positions of the double bonds is poorly understood. In this work, we shed light on this matter through atomic-scale molecular dynamics simulations of eight different one-component lipid bilayers comprised of PCs with 18 carbons in their acyl chains. By introducing a single double bond in each acyl chain and varying its position in a systematic manner, we elucidate the effects of a double bond on various membrane properties. Studies in the fluid phase show that a number of membrane properties depend on the double bond position. In particular, when the double bond in an acyl chain is located close to the membrane-water interface, the area per lipid is considerably larger than that found for a saturated lipid. Further, when the double bond is shifted from the interfacial region toward membrane center, the area per lipid is observed to increase and have a maximum when the double bond is in the middle of the chain. Beyond this point, the surface area decreases systematically as the double bond approaches membrane center. These changes in area per lipid are accompanied by corresponding changes in membrane thickness and ordering of the chains. Further changes are observed in the tilt angles of the chains, membrane hydration together with changes in the number of gauche conformations, and direct head group interactions. All of these effects can be associated with changes in acyl chain conformations and local effects of the double bond on the packing of the surrounding atoms.     

Molecular dynamics simulations of cardiolipin bilayers

Cardiolipin is a key lipid component in the inner mitochondrial membrane, where the lipid is involved in energy production, cristae structure, and mechanisms in the apoptotic pathway. In this article we used molecular dynamics computer simulations to investigate cardiolipin and its effect on the structure of lipid bilayers. Three cardiolipin/POPC bilayers with different lipid compositions were simulated: 100, 9.2, and 0% cardiolipin. We found strong association of sodium counterions to the carbonyl groups of both lipid types, leaving in the case of 9.2% cardiolipin virtually no ions in the aqueous compartment. Although binding occurred primarily at the carbonyl position, there was a preference to bind to the carbonyl groups of cardiolipin. Ion binding and the small headgroup of cardiolipin gave a strong ordering of the hydrocarbon chains. We found significant effects in the water dipole orientation and water dipole potential which can compensate for the electrostatic repulsion that otherwise should force charged lipids apart. Several parameters relevant for the molecular structure of cardiolipin were calculated and compared with results from analyses of coarse-grained simulations and available X-ray structural data.     

Selective assembly of photosynthetic antenna proteins into a domain-structured lipid bilayer for the construction of artificial photosynthetic antenna systems: structural analysis of the assembly using surface plasmon resonance and atomic force microscopy

Two types of photosynthetic membrane proteins, the peripheral antenna complex (LH2) and the core antenna/reaction center complex (LH1-RC), play an essential role in the primary process of purple bacterial photosynthesis, that is, capturing light energy, transferring it to the RC where it is used in subsequent charge separation. Establishment of experimental platforms is required to understand the function of the supramolecular assembly of LH2 and LH1-RC molecules into arrays. In this study, we assembled LH2 and LH1-RC arrays into domain-structured planar lipid bilayers placed on a coverglass using stepwise combinations of vesicle-to-planar membrane formation and vesicle fusion methods. First, it was shown that assembly of LH2 and LH1-RC in planar lipid bilayers, through vesicle-to-planar membrane formation, could be confirmed by absorption spectroscopy and high resolution atomic force microscopy (AFM). Second, formation of a planar membrane incorporating LH2 molecules made by the vesicle fusion method was corroborated by AFM together with quantitative analysis by surface plasmon resonance (SPR). By combining planar membrane formation and vesicle fusion, in a stepwise manner, LH2 and LH1-RC were successfully organized in the domain-structured planar lipid membrane. This methodology for construction of LH2/LH1-RC assemblies will be a useful experimental platform with which to investigate energy transfer from LH2 to LH1-RC where the relative arrangement of these two complexes can be controlled.     

Modulating membrane properties: the effect of trehalose and cholesterol on a phospholipid bilayer

The protective properties of trehalose on cholesterol-containing lipid dipalmitoylphosphatidylcholine (DPPC) bilayers are studied through molecular simulations. The ability of the disaccharide to interact with the phospholipid headgroups and stabilize the membrane persists even at high cholesterol concentrations and restricts some of the changes to the structure that would otherwise be imposed by cholesterol molecules. Predictions of bilayer properties such as area per lipid, tail ordering, and chain conformation support the notion that the disaccharide decreases the main melting transition in these multicomponent model membranes, which correspond more closely to common biological systems than pure bilayers. Molecular simulations indicate that the membrane dynamics are slowed considerably by the presence of trehalose, indicating that high sugar concentrations would serve to avert possible phase separations that could arise in mixed phospholipid systems. Various time correlation functions suggest that the character of the modifications in lipid dynamics induced by trehalose and cholesterol is different in the hydrophilic and hydrophobic regions of the membrane.     

Atomic force microscopy force mapping in the study of supported lipid bilayers

Investigating the structural and mechanical properties of lipid bilayer membrane systems is vital in elucidating their biological function. One route to directly correlate the morphology of phase-segregated membranes with their indentation and rupture mechanics is the collection of atomic force microscopy (AFM) force maps. These force maps, while containing rich mechanical information, require lengthy processing time due to the large number of force curves needed to attain a high spatial resolution. A force curve analysis toolset was created to perform data extraction, calculation and reporting specifically in studying lipid membrane morphology and mechanical stability. The procedure was automated to allow for high-throughput processing of force maps with greatly reduced processing time. The resulting program was successfully used in systematically analyzing a number of supported lipid membrane systems in the investigation of their structure and nanomechanics.     

Impact of Strong and Weak Lipid–Protein Interactions on the Structure of a Lipid Bilayer on a Gold Electrode Surface

A lipid bilayer deposited on an electrode surface can serve as a benchmark system to investigate lipid–protein interactions in the presence of physiological electric fields. Recoverin and myelin‐associated glycoprotein (MAG) are used to study the impact of strong and weak protein–lipid interactions on the structure of model lipid bilayers, respectively. The structural changes in lipid bilayers are followed using electrochemical polarization modulation infrared reflection–absorption spectroscopy (PM IRRAS). Recoverin contains a myristoyl group that anchors in the hydrophobic part of a cell membrane. Insertion of the protein into the 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphatidylcholine (DMPC)–cholesterol lipid bilayer leads to an increase in the capacitance of the lipid film adsorbed on a gold electrode surface. The stability and kinetics of the electric‐field‐driven adsorption–desorption process are not affected by the interaction with protein. Upon interaction with recoverin, the hydrophobic hydrocarbon chains become less ordered. The polar head groups are separated from each other, which allows for recoverin association in the membrane. MAG is known to interact with glycolipids present on the surface of a cell membrane. Upon probing the interaction of the DMPC–cholesterol–glycolipid bilayer with MAG a slight decrease in the capacity of the adsorbed lipid film is observed. The stability of the lipid bilayer increases towards negative potentials. At the molecular scale this interaction results in minor changes in the structure of the lipid bilayer. MAG causes small ordering in the hydrocarbon chains region and an increase in the hydration of the polar head groups. Combining an electrochemical approach with a structure‐sensitive technique, such as PM IRRAS, is a powerful tool to follow small but significant changes in the structure of a supramolecular assembly.     

Temperature‐Dependent Atomic Models of Detergent Micelles Refined against Small‐Angle X‐Ray Scattering Data

Surfactants have found a wide range of industrial and scientific applications. In particular, detergent micelles are used as lipid membrane mimics to solubilize membrane proteins for functional and structural characterization. However, an atomic‐level understanding of surfactants remains limited because many experiments provide only low‐resolution structural information on surfactant aggregates. In this work, small‐angle X‐ray scattering is combined with molecular dynamics simulations to derive fully atomic models of two maltoside micelles at temperatures between 10 °C and 70 °C. The micelles take the shape of general tri‐axial ellipsoids and decrease in size and aggregation number with increasing temperature. Density profiles of hydrophobic groups and water along the three principal axes reveal that the minor micelle axis closely mimics lipid membranes. The results suggest that coupling atomic simulations with low‐resolution data allows the structural characterization of surfactant aggregates.