Bilayer Location Research Articles

Location of dopamine in lipid bilayers and its relevance to neuromodulator function

Dopamine (DA) is a neurotransmitter that also acts as a neuromodulator, with both functions being essential to brain function. Here, we present the first experimental measurement of DA location in lipid bilayers using x-ray diffuse scattering, solid-state deuterium NMR, and electron paramagnetic resonance. We find that the association of DA with lipid headgroups as seen in electron density profiles leads to an increase of intermembrane repulsion most likely due to electrostatic charging. DA location in the lipid headgroup region also leads to an increase of the cross-sectional area per lipid without affecting the bending rigidity significantly. The order parameters measured by solid-state deuterium NMR decrease in the presence of DA for the acyl chains of PC and PS lipids, consistent with an increase in the area per lipid due to DA. Most importantly, these results support the hypothesis that three-dimensional diffusion of DA to target membranes could be followed by relatively more efficient two-dimensional diffusion to receptors within those membranes.

Interactions of Novel Phosphonium Dye with Lipid Bilayers: A Molecular Dynamics Study

In the present work the 100-ns molecular dynamics simulations (MD) were performed in the CHARMM36m force field using the GROMACS package to estimate the bilayer location and mechanisms of the interaction between the novel phosphonium dye TDV and the model lipid membranes composed of the phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) or/and anionic phospholipid cardiolipin (CL). Varying the dye initial position relative to the membrane midplane, the dye relative orientation and the charge state of the TDV molecule it was found that the one charge form of TDV, which was initially translated to a distance of 20 Å from the membrane midplane along the bilayer normal, readily penetrates deeper into the membrane interior and remains within the lipid bilayer during the entire simulation time. It was revealed that the probe partitioning into the model membranes was accompanied by the reorientation of TDV molecule from perpendicular to nearly parallel to the membrane surface. The analysis of the MD simulation results showed that the lipid bilayer partitioning and location of the one charge form of TDV depend on the membrane composition. The dye binds more rapidly to the neat PC bilayer than to CL- and Chol-containing model membranes. It was found that in the neat PC and CL-containing membranes the one charge TDV resides at the level of carbonyl groups of lipids (the distances ~ 1.1 nm, 1.2 nm and 1.3 nm from the bilayer center for the PC, CL10 and CL20 lipid membranes, respectively), whereas in the Chol-containing membranes the probe is located at the level of glycerol moiety (~ 1.5 nm and 1.6 nm for the Chol30 and CL10/Chol30 lipid membranes, respectively). It was demonstrated that the dye partitioning into the lipid bilayer does not affect the membrane structural properties.

Study of Resveratrol's Interaction with Planar Lipid Models: Insights into Its Location in Lipid Bilayers.

Resveratrol, a polyphenolic molecule found in edible fruits and vegetables, shows a wide range of beneficial effects on human health, including anti-microbial, anti-inflammatory, anti-cancer, and anti-aging properties. Due to its poor water solubility and high liposome-water partition coefficient, the biomembrane seems to be the main target of resveratrol, although the mode of interaction with membrane lipids and its location within the cell membrane are still unclear. In this study, using electrophysiological measurements, we study the interaction of resveratrol with planar lipid membranes (PLMs) of different composition. We found that resveratrol incorporates into palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:Ch PLMs and forms conductive units unlike those found in dioleoyl-phosphatidylserine (DOPS):dioleoyl-phosphatidylethanolamine (DOPE) PLMs. The variation of the biophysical parameters of PLMs in the presence of resveratrol provides information on its location within a lipid double layer, thus contributing to an understanding of its mechanism of action.

Molecular Dynamics Simulation of the Interaction Between Benzanthrone Dye and Model Lipid Membranes

The benzanthrone fluorescent dyes are known as environmentally-sensitive reporters for exploring the physicochemical properties and structural alterations of lipid membranes. In the present work the 100-ns molecular dynamics simulation (MD) was used to characterize the bilayer location and the nature of interactions between the benzanthrone fluorescent dye ABM and the model lipid membranes composed of the zwitterionic lipid phosphatidylcholine (PC) and its mixtures with the anionic lipid phosphatidylglycerol (PG20) and sterol cholesterol (Chol30). The MD simulations were performed in the CHARMM36m force field using the GROMACS package. The ABM molecule, which was initially placed at a distance of 30 Å from the midplane of the lipid bilayer, after 10 ns of simulation was found to be completely incorporated into the membrane interior and remained within the lipid bilayer for the rest of the simulation time. The analysis of the MD simulation results showed that the lipid bilayer location of the benzanthrone dye ABM depends on the membrane composition, with the distance from bilayer center being gradually shifted from 0.78 nm in the neat PC bilayer to 0.95 nm and 1.5 nm in the PG- and Chol-containing membranes, respectively. In addition, the partitioning of the ABM into the neat PC bilayer was followed by the probe translocation from the outer membrane leaflet to the inner one. A separate series of MD simulations was aimed at examining the ABM influence on the lipid bilayer structure. It was found that ABM partitioning into the lipid bilayers of various composition has no significant effect on the orientation of the fatty acid chains and leads only to a small increase of the deuterium order parameter for the carbon atoms 5-to-8 in the sn-2 acyl chains of the neat PC membranes. In addition, the interaction of the ABM with the model lipid membranes caused the slight decrease of the surface area per lipid pointing to the slight increase of the packing density of lipid molecules in the presence of ABM. The results obtained provide a basis for deeper understanding of the membrane interactions of benzanthrone dyes and may be useful for the design of the novel fluorescent probes for membrane studies.

Molecular Dynamics Simulations Reveal a Possible Membrane‐Involved Microkinetic Mechanism for Long‐Lasting Action of Salmeterol, a β2‐Adrenergic Receptor Agonist

ObjectiveThe exact mechanisms underlying the longer duration of action and capacity to “reassert” airway smooth muscle relaxation in vitro despite repeated washing, of several long‐acting β2‐adrenergic receptor (β2‐AR) agonists such as salmeterol remain unresolved. Recent experimental studies suggest that the “microkinetic model” in which membrane acts as a depot enriching local drug concentration around the receptor adequately explains the long‐lasting effects. Specifically, the 700‐fold higher potency of salmeterol to β2‐AR in comparison to the structurally related agonists was found to be majorly due to its interactions with the membrane. The ‘reduction of dimensionality’ effect whereby ligands move on or within the membrane by 2D lateral diffusion could likely produce further enhancement of drug association rates and corresponding affinity values. These studies also suggested that the “exosite” described before probably reflects the phospholipid membrane rather than specific residues on the receptor. Most recently, a crystal structure of salmeterol bound to the active β2‐AR revealed an exosite for its aryloxyalkyl tail at the extracellular vestibule of the receptor. However, the association and dissociation pathways through which salmeterol access and leave the binding site is unknown. In addition, the possibility of lipophilic salmeterol reaching the binding site through transmembrane helices from the surrounding membrane environment is compelling and warrants thorough investigation, especially with the availability of the bound crystal structure. Here, we present an extensive computational study investigating the plausible lipid‐mediated association and dissociation pathways of salmeterol.MethodsThe most favorable bilayer location, orientation, and conformation of salmeterol were investigated using a 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) model membrane with 10% cholesterol by steered molecular dynamics (MD) and Umbrella Sampling simulations, using NAMD2.12 software. The dissociation and association pathways of salmeterol from the bound β2‐AR crystal structure (PDB ID 6CSY) were investigated by: 1) steered MD, 2) temperature accelerated MD, and 3) funnel metadynamics simulations. The protein, POPC lipids, and salmeterol were parameterized using the CHARMM36 and CGenFF force fields respectively.ResultsThe results thus far, reveal that the dissociation of salmeterol from its bound form proceeds through the extracellular side of the receptor, with a transient stay in the aqueous bulk before finally partitioning into the membrane (near the β2‐AR) in its energetically favorable bilayer depth (~15 Å from the bilayer center) determined by our partitioning studies. The specific interactions of the ligand with the receptor residues along the binding pathways and associated free energies were analyzed.ConclusionsComputational results suggest that the ‘microkinetic” mechanism attributed to the high membrane affinity of salmeterol may play a concerted role with its exosite binding towards the unique pharmacological properties.Support or Funding InformationNIH – R15GM131293This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Orientation of nitro-group governs the fluorescence lifetime of nitrobenzoxadiazole (NBD)-labeled lipids in lipid bilayers.

Nitrobenzoxadiazole (NBD) labeled lipids are popular fluorescent probes of membrane structure and dynamics, and have been widely used in both model systems and living cells. Irrespective of attachment to the lipid head group or hydrocarbon chains, the NBD fluorophore generally adopts a transverse bilayer location near the host lipid carbonyl/glycerol moieties. Still, considerable variability is observed in the measured fluorescence lifetimes, indicating that overall fluorophore location is not the determinant of NBD fluorescence properties. Combining fluorescence experiments and molecular dynamics simulations, we show that for two almost identical NBD probes, significant differences in fluorophore orientation and fluorescence lifetime are observed. Integrating these findings with literature data, we demonstrate a correlation between NBD orientation and fluorescence lifetime. The latter is longer when the NBD nitro group is predominantly oriented towards the bilayer interior, compared to probes for which it points to the water medium.

Cholesterol's location in lipid bilayers

It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol's location in lipid bilayers has been studied extensively, and it has been shown – at least in some bilayers – to align differently from its canonical upright orientation, where its hydroxyl group is in the vicinity of the lipid–water interface. In this article we review recent works describing cholesterol's location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies.

Lipid-Linked Oligosaccharides in Membranes Sample Conformations That Facilitate Binding to Oligosaccharyltransferase

Lipid-linked oligosaccharides (LLOs) are the substrates of oligosaccharyltransferase (OST), the enzyme that catalyzes the en bloc transfer of the oligosaccharide onto the acceptor asparagine of nascent proteins during the process of N-glycosylation. To explore LLOs’ preferred location, orientation, structure, and dynamics in membrane bilayers of three different lipid types (dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine), we have modeled and simulated both eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol) and bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol) LLOs, which are composed of an isoprenoid moiety and an oligosaccharide, linked by pyrophosphate. The simulations show no strong impact of different bilayer hydrophobic thicknesses on the overall orientation, structure, and dynamics of the isoprenoid moiety and the oligosaccharide. The pyrophosphate group stays in the bilayer head group region. The isoprenoid moiety shows high flexibility inside the bilayer hydrophobic core, suggesting its potential role as a tentacle to search for OST. The oligosaccharide conformation and dynamics are similar to those in solution, but there are preferred interactions between the oligosaccharide and the bilayer interface, which leads to LLO sugar orientations parallel to the bilayer surface. Molecular docking of the bacterial LLO to a bacterial OST suggests that such orientations can enhance binding of LLOs to OST.

Structure-based prediction of drug distribution across the headgroup and core strata of a phospholipid bilayer using surrogate phases.

Solvation of drugs in the core (C) and headgroup (H) strata of phospholipid bilayers affects their physiological transport rates and accumulation. These characteristics, especially a complete drug distribution profile across the bilayer strata, are tedious to obtain experimentally, to the point that even simplified preferred locations are only available for a few dozen compounds. Recently, we showed that the partition coefficient (P) values in the system of hydrated diacetyl phosphatidylcholine (DAcPC) and n-hexadecane (C16), as surrogates of the H- and C-strata of the bilayer composed of the most abundant mammalian phospholipid, PC, agree well with the preferred bilayer location of compounds. High P values are typical for lipophiles accumulating in the core, and low P values are characteristic of cephalophiles preferring the headgroups. This simple pattern does not hold for most compounds, which usually have more even distribution and may also accumulate at the H/C interface. To model complete distribution, the correlates of solvation energies are needed for each drug state in the bilayer: (1) for the H-stratum it is the DAcPC/W P value, calculated as the ratio of the C16/W and C16/DAcPC (W for water) P values; (2) for the C-stratum, the C16/W P value; (3) for the H/C interface, the P values for all plausible molecular poses are characterized using the fragment DAcPC/W and C16/W solvation parameters for the parts of the molecule embedded in the H- and C-strata, respectively. The correlates, each scaled by two Collander coefficients, were used in a nonlinear, mass-balance based model of intrabilayer distribution, which was applied to the easily measurable overall P values of compounds in the DMPC (M = myristoyl) bilayers and monolayers as the dependent variables. The calibrated model for 107 neutral compounds explains 94% of experimental variance, achieves similar cross-validation levels, and agrees well with the nontrivial, experimentally determined bilayer locations for 27 compounds. The resulting structure-based prediction system for intrabilayer distribution will facilitate more realistic modeling of passive transport and drug interactions with those integral membrane proteins, which have the binding sites located in the bilayer, such as some enzymes, influx and efflux transporters, and receptors. If only overall bilayer accumulation is of interest, the 1-octanol/W P values suffice to model the studied set.

Dimyristoyl Phosphatidylcholine: A Remarkable Exception to α-Tocopherol’s Membrane Presence

Using data obtained from different physical techniques (i.e., neutron diffraction, NMR and UV spectroscopy), we present evidence which explains some of the conflicting and inexplicable data found in the literature regarding α-tocopherol's (aToc's) behavior in dimyristoyl phosphatidylcholine (di-14:0PC) bilayers. Without exception, the data point to aToc's active chromanol moiety residing deep in the hydrophobic core of di-14:0PC bilayers, a location that is in stark contrast to aToc's location in other PC bilayers. Our result is a clear example of the importance of lipid species diversity in biological membranes and importantly, it suggests that measurements of aToc's oxidation kinetics, and its associated byproducts observed in di-14:0PC bilayers, should be reexamined, this time taking into account its noncanonical location in this bilayer.

Structural determinants of drug partitioning in surrogates of phosphatidylcholine bilayer strata.

The knowledge of drug concentrations in bilayer headgroups, core, and at the interface between them is a prerequisite for quantitative modeling of drug interactions with many membrane-bound transporters, metabolizing enzymes and receptors, which have the binding sites located in the bilayer. This knowledge also helps understand the rates of trans-bilayer transport because balanced interactions of drugs with the bilayer strata lead to high rates, while excessive affinities for any stratum cause a slowdown. Experimental determination of bilayer location is so tedious and costly that the data are only available for some fifty compounds. To extrapolate these valuable results to more compounds at a higher throughput, surrogate phases have been used to obtain correlates of the drug affinities for individual strata. We introduced a novel system, consisting of a diacetyl phosphatidylcholine (DAcPC) solution with the water content of the fluid bilayer as the headgroup surrogate and n-hexadecane (C16) representing the core. The C16/DAcPC partition coefficients were measured for 113 selected compounds, containing structural fragments that are frequently occurring in approved drugs. The data were deconvoluted into the ClogP-based fragment solvation characteristics and processed using a solvatochromic correlation. Increased H-bond donor ability and excess molar refractivity of compounds promote solvation in the DAcPC phase as compared to bulk water, contrary to H-bond acceptor ability, dipolarity/polarizability, and volume. The results show that aromates have more balanced distribution in bilayer strata, and thus faster trans-bilayer transport, than similar alkanes. This observation is in accordance with the frequent occurrence of aromatic rings in approved drugs and with the role of rigidity of drug molecules in promoting intestinal absorption. Bilayer locations, predicted using the C16/DAcPC system, are in excellent agreement with available experimental data, in contrast to other surrogate systems.

Reaction Field Analysis and Lipid Bilayer Location for Lipophilic Fluorophores

Environment polarity can cause changes in absorbance or emission maxima, for a given fluorophore. This is termed solvatochromism. In this study semiempirical models of solvatochromic shifts are used to predict their lipid bilayer location. Four reaction field models are analyzed and compared, to provide the most accurate prediction of fluorophore solvatochromic shifts using a modified version of the Lippert equation. For curcumin, the reaction field of Block and Walker gave the strongest agreement between experimental and predicted values (r = 0.978, p < 0.0001). For aluminum phthalocyanine disulfonic acid (AlPcS2), the reaction field of Wertheim, based on statistical mechanics, gave the best agreement (r = 0.951, p = 0.001) only when dispersion forces and solute polarizability are considered. The results of these models are correlated to the Dimroth-Reichardt ET(30) solvent polarity scale used by Frimer and colleagues. Using the model predicted values, curcumin is estimated to be 1-1.2 nm from the phospholipid-water interface, in the acyl chain region of the lipid bilayer. AlPcS2 is predicted to be 0.7-0.9 nm from the interface, at the fatty acid carbonyl. This investigation provides semiempirical methods to efficiently link fluorophore solvatochromic shifts to a location in the lipid bilayer via reaction field models.

Vibrational, calorimetric, and molecular conformational study on calcein interaction with model lipid membrane

Nanoliposomes are commonly used as a carrier in controlled release drug delivery systems. Controlled release formulations can be used to reduce the amount of drug necessary to cause the same therapeutic effect in patients. One of the most noticeable factors in release profiles is the strength of the drug-carrier interaction. To adjust the pharmacokinetic and pharmacodynamic properties of therapeutic agents, it is necessary to optimize the drug-carrier interaction. To get a better understanding of this interaction, large unilamellar liposomes containing calcein were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1,2-palmitoyl-sn-glycero-3-phosphocholine, and a mixture of them; calcein was chosen as a model polar molecule of biological interest. The thermodynamic changes induced by calcein and its location in lipid bilayers were determined by differential scanning calorimetry and Raman spectroscopy, respectively. The results reveal that calcein has no significant influence on thermotropic properties of the lipid membrane, but causing the abolition of pre-transition. The decreasing of the pre-transition can be ascribed to the presence of calcein near the hydrophilic cooperative zone of the bilayer. The change in intensity of the Raman peaks represents the interaction of calcein with choline head groups. Moreover, the impact of calcein on phosphoglyceride Langmuir layers spread at the air–water interface was studied using surface pressure-area and surface potential-area isotherms, as well as polarization-modulation infrared reflection–absorption spectroscopy and Brewster angle microscopy. The results obtained indicate that calcein introduce no major modification on the systems prepared with pure lipids.

Structural determinants of drug partitioning in n-hexadecane/water system.

Surrogate phases have been widely used as correlates for modeling transport and partitioning of drugs in biological systems, taking advantage of chemical similarity between the surrogate and the phospholipid bilayer as the elementary unit of biological phases, which is responsible for most of the transport and partitioning. Solvation in strata of the phospholipid bilayer is an important drug characteristic because it affects the rates of absorption and distribution, as well as the interactions with the membrane proteins having the binding sites located inside the bilayer. The bilayer core can be emulated by n-hexadecane (C16), and the headgroup stratum is often considered a hydrophilic phase because of the high water content. Therefore, we tested the hypothesis that the C16/water partition coefficients (P) can predict the bilayer locations of drugs and other small molecules better than other surrogate systems. Altogether 514 PC16/W values for nonionizable (458) and completely ionized (56) compounds were collected from the literature or measured, when necessary. With the intent to create a fragment-based prediction system, the PC16/W values were factorized into the fragment solvation parameters (f) and correction factors based on the ClogP fragmentation scheme. A script for the PC16/W prediction using the ClogP output is provided. To further expand the prediction system and reveal solvation differences, the fC16/W values were correlated with their more widely available counterparts for the 1-octanol/water system (O/W) using solvatochromic parameters. The analysis for 50 compounds with known bilayer location shows that the available and predicted PC16/W and PO/W values alone or the PC16/O values representing their ratio do not satisfactorily predict the preference for drug accumulation in bilayer strata. These observations indicate that the headgroups stratum, albeit well hydrated, does not have solvation characteristics similar to water and is also poorly described by the O/W partition characteristics.

Structure-Function Relationships of Antimicrobial Piscidins 1 and 3 bound to Cholesterol-Containing Lipid Membranes

Isolated in the mast cells of hybrid-striped sea bass, piscidin 1 (p1) and piscidin 3 (p3) are antimicrobial, cationic, and amphipathic peptides that have demonstrated broad-spectrum activity against bacteria, fungi, viruses, and cancer cells. Both p1 and p3 adopt an alpha-helical structure when bound to phospholipid membranes. p1 is the more active of the two isoforms, exhibiting higher lytic activity on different bacterial strains, as well as erythrocytes. This research uses various biophysical methods to investigate the differences in the backbone structure and bilayer location of piscidin bound to different lipid bilayers of biological relevance.To mimic the composition of human erythrocytes, a mixture of 4:1 palmitoyl-oleoyl-phosphatidylcholine (POPC):cholesterol (CHL) at pH 7.4 was used. Oriented bilayer samples prepared with 1:40 peptide:lipid were analyzed using solid-state NMR yielding 15N-1H dipolar couplings as well as 15N amide chemical shifts. These data were used to compute a high-resolution atomic-level structure of p1 and p3 bound to zwitterionic bilayers. The peptide's backbone structure is largely conserved across various bacterial and mammalian membrane mimics, suggesting that the difference in a peptide's activity on various membranes is more reliant on the peptide's side-chain and its position within the membrane.The structural results were supplemented with dye leakage assays in order to probe the peptide's activity on lipid vesicles, mimicking bacterial and mammalian cells. These results lend insight into how specific changes in peptide's structure may result in varying activity on different membrane systems. Molecular dynamics (MD) simulations were used to predict the peptide's bilayer location and depth of insertion. Membrane thinning, an event known to precede pore formation, was shown using MD. Overall, these experiments help obtain principles to design novel antibiotic pharmaceuticals with low hemolytic effects and high lytic activity on bacteria.

Atomic-Resolution Three Dimensional Structures and Membrane Locations of Antimicrobial Piscidin 1 and Piscidin 3 in Aligned Lipid Bilayers: A Solid-State NMR and Molecular Dynamics Investigation

Piscidin, an amphipathic cationic antimicrobial peptide (AMP) active against a broad range of pathogens including multidrug-resistant bacteria and HIV-1, belongs to a large family of vital host-defense peptides that interact, at least initially, with negatively-charged microbial membranes in order to perform their function. While two piscidin isoforms, piscidin 1 (p1) and piscidin 3 (p3), are highly homologous, they display unequal antimicrobial and hemolytic effects. As a way to identify factors optimizing specific molecular interactions directly related to their mode of membrane activity, we have investigated p1 and p3 bound to lipid membranes that mimic bacterial membranes. Previously, we used solid-state NMR on 15N-labeled peptides to demonstrate that membrane-bound p1 and p3 adopt an alpha-helical structure and lie in the plane of hydrated lipid bilayers where they experience fast dynamics. Our recent analysis of two-dimensional solid-state NMR data has lead to the first atomic resolution three-dimensional backbone structures of p1 and p3 bound to aligned lipid bilayers. Structural calculations based on the NMR data and molecular dynamics simulations have been performed to yield a refined structure and membrane location for each peptide. We will explain how our atomic-level investigation of the structure, dynamics, and bilayer location of piscidin provides new insights into its mode of action and therefore allows us better to understand how AMPs disrupt bacterial membranes and induce cell death. The long term goal is to derive common principles that could facilitate the design of pharmaceuticals with enhanced antibacterial activity and lower toxicity on mammalian cells.

N- and O-methylation of sphingomyelin markedly affects its membrane properties and interactions with cholesterol

We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability (Tm was lowered by ~7°C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers.

Protein Folding in Membranes: Insights from Neutron Diffraction Studies of a Membrane β-Sheet Oligomer

Studies of the assembly of the hexapeptide Acetyl-Trp-Leu 5 (AcWL 5) into β-sheets in membranes have provided insights into membrane protein folding. Yet, the exact structure of the oligomer in the lipid bilayer is unknown. Here we use neutron diffraction to study the disposition of the peptides in bilayers. We find that pairs of adjacent deuterium-labeled leucines have no well-defined peak or dip in the transmembrane distribution profiles, indicative of heterogeneity in the depth of membrane insertion. At the same time, the monomeric homolog AcWL 4 exhibits a homogeneous, well-defined, interfacial location in neutron diffraction experiments. Thus, although the bilayer location of monomeric AcWL 4 is determined by hydrophobicity matching or complementarity within the bilayer, the AcWL 5 molecules in the oligomer are positioned at different depths within the bilayer because they assemble into a staggered transmembrane β-sheet. The AcWL 5 assembly is dominated by protein-protein interactions rather than hydrophobic complementarity. These results have implications for the structure and folding of proteins in their native membrane environment and highlight the importance of the interplay between hydrophobic complementarity and protein-protein interactions in determining the structure of membrane proteins.

Cytochrome c Interaction with Cardiolipin/Phosphatidylcholine Model Membranes: Effect of Cardiolipin Protonation

Resonance energy transfer between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptor has been employed to explore the protein binding to model membranes, composed of phosphatidylcholine and cardiolipin (CL). The existence of two types of protein-lipid complexes has been hypothesized where either deprotonated or partially protonated CL molecules are responsible for cyt c attachment to bilayer surface. To quantitatively describe cyt c membrane binding, the adsorption model based on scaled particle and double layer theories has been employed, with potential-dependent association constants being treated as a function of acidic phospholipid mole fraction, degree of CL protonation, ionic strength, and surface coverage. Multiple arrays of resonance energy transfer data obtained under conditions of varying pH, ionic strength, CL content, and protein/lipid molar ratio have been analyzed in terms of the model of energy transfer in two-dimensional systems combined with the adsorption model allowing for area exclusion and electrostatic effects. The set of recovered model parameters included effective protein charge, intrinsic association constants, and heme distance from the bilayer midplane for both types of protein-lipid complexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close to that characteristic of the inner mitochondrial membrane), the binding equilibrium dramatically shifted toward cyt c association with partially protonated CL species. The estimates of heme distance from bilayer center suggest shallow bilayer location of cyt c at physiological pH, whereas at pH below 6.0, the protein tends to insert into membrane core.

Partitioning of organic compounds in phases imitating the headgroup and core regions of phospholipid bilayers.

Solvation free energies of drugs, peptides, and other small molecules in the core and headgroup regions of phospholipid bilayers determine their conformations, accumulation, and transport properties. The transfer free energy includes the energy terms for the formation of a cavity for the solute, the interactions of the solute with phospholipids, electrostatic interactions of the solute with the membrane, and dipole potentials and entropy terms. The interaction energies with phospholipids can be estimated by correlating the partitioning in surrogate solvent systems and in the bilayer. As the headgroup surrogate, we use diacetylphosphatidylcholine (DAcPC), the acetylated headgroup of the most abundant mammalian phospholipid, phosphatidylcholine, which forms a homogeneous solution with acceptable viscosity when mixed with water in ratios similar to those in the fully hydrated bilayer. The two-phase system of n-hexadecane (C16) as the core surrogate and hydrated DAcPC was used to monitor partitioning of 16 nonionizable compounds. On the bilogarithmic scale, the C16/DAcPC partition coefficients correlate neither with those in the C16/water and 1-octanol/water systems nor with their difference, which is frequently used as a parameter of hydrogen bonding for prediction of the bilayer location of the solutes. The C16/DAcPC system provides a satisfactory emulation of the solvation properties of the bilayer regions, as reflected in correct predictions of the bilayer location for those of the studied chemicals, for which this information is available.