The interaction and orientation of Peptide KL4 in model membranes.

We report on the orientation and location of synthetic pulmonary surfactant peptide KL4, (KLLLL)4K, in model lipid membranes. The partitioning depths of selectively deuterated leucine residues within KL4 were determined in DPPC:POPG (4:1) and POPC:POPG (4:1) bilayers by oriented neutron diffraction. These measurements were combined with an NMR-generated model of the peptide structure to determine the orientation and partitioning of the peptide at the lipid-water interface. The results demonstrate KL4 adopting an orientation that interacts with a single membrane leaflet. These observations are consistent with past 2H NMR and EPR studies (Antharam et al., 2009; Turner et al., 2014).

The structures of polyunsaturated lipid bilayers by joint refinement of neutron and X-ray scattering data.

We present the detailed structural analysis of polyunsaturated fatty acid-containing phospholipids namely, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC). A newly developed molecular dynamics (MD) simulation parsing scheme for lipids containing fatty acids with multiple double bonds was implemented into the scattering density profile (SDP) model to simultaneously refine differently contrasted neutron and X-ray scattering data. SDP analyses of scattering data at 30 °C yielded lipid areas of 71.1 Å2 and 70.4 Å2 for PDPC and SDPC bilayers, respectively, and a model free analysis of PDPC at 30 °C resulted in a lipid area of 72 Å2. In addition to bilayer structural parameters, using area-constrained MD simulations we determined the area compressibility modulus, KA, to be 246.4 mN/m, a value similar to other neutral phospholipids.

Vitamin E-inspired multi-scale imaging agent.

The production and use of multi-modal imaging agents is on the rise. The vast majority of these imaging agents are limited to a single length scale for the agent (e.g. tissues only), which is typically at the organ or tissue scale. This work explores the synthesis of such an imaging agent and discusses the applications of our vitamin E-inspired multi-modal and multi-length scale imaging agents TB-Toc ((S,E)-5,5-difluoro-7-(2-(5-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl) methyl) thiophen-2-yl) vinyl)-9-methyl-5H-dipyrrolo-[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide). We investigate the toxicity of TB-Toc along with the starting materials and lipid based delivery vehicle in mouse myoblasts and fibroblasts. Further we investigate the uptake of TB-Toc delivered to cultured cells in both solvent and liposomes. TB-Toc has low toxicity, and no change in cell viability was observed up to concentrations of 10 mM. TB-Toc shows time-dependent cellular uptake that is complete in about 30 min. This work is the first step in demonstrating our vitamin E derivatives are viable multi-modal and length scale diagnostic tools.

Testing High Concentrations of Membrane Active Antibiotic Chlorhexidine Via Computational Titration and Calorimetry.

Coarse-grained strategies for membrane simulations are designed to increase efficiency for larger and more complex molecular dynamics simulations. For membrane active antibiotics, the concentration dependence of their action presents a tremendous challenge in simulation scale. In this study, we examine the effects of concentration for the popular membrane active antibacterial drug chlorhexidine. It presents an interesting biophysical modeling test, where from experimental experience we know that model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) can absorb very high quantities of the drug without disruption. We construct a coarse-grained model of chlorhexidine in three different charged states and compare to previous all-atom simulations and new experiments. Using large, long-time, and unbiased simulations of chlorhexidine inserting into the lipid bilayer, we find little changes to the functional structure of a DMPC membrane up through concentrations of 15:100 drug:lipid, where the slowing rate of continued insertion tests the capabilities of even this coarse-grained approach. We validate our simulations with computational calorimetry measurements, and show that they agree with new experimental data from differential scanning calorimetry.

Fluctuations in Hertz chains at equilibrium.

We examine the long-term behavior of nonintegrable, energy-conserved, one-dimensional systems of macroscopic grains interacting via a contact-only generalized Hertz potential and held between stationary walls. Such systems can be set up to have no phononic background excitation and represent examples of a sonic vacuum. Existing dynamical studies showed the absence of energy equipartitioning in such systems, hence their long-term dynamics was described as quasiequilibrium. Here we show that these systems do in fact reach thermal equilibrium at sufficiently long times, as indicated by the calculated heat capacity. As a by-product, we show how fluctuations of system quantities, and thus the distribution functions, are influenced by the Hertz potential. In particular, the variance of the system's kinetic energy probability density function is reduced by a factor related to the contact potential.

Vitamin E Circular Dichroism Studies: Insights into Conformational Changes Induced by the Solvent's Polarity.

We used circular dichroism (CD) to study differences in CD spectra between α-, δ-, and methylated-α-tocopherol in solvents with different polarities. CD spectra of the different tocopherol structures differ from each other in intensity and peak locations, which can be attributed to chromanol substitution and the ability to form hydrogen bonds. In addition, each structure was examined in different polarity solvents using the Reichardt index-a measure of the solvent's ionizing ability, and a direct measurement of solvent-solute interactions. Differences across solvents indicate that hydrogen bonding is a key contributor to CD spectra at 200 nm. These results are a first step in examining the hydrogen bonding abilities of vitamin E in a lipid bilayer.

Lipid bilayer thickness determines cholesterol's location in model membranes.

Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of different lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. These results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.

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.

A MARTINI extension for Pseudomonas aeruginosa PAO1 lipopolysaccharide.

We report a course-grained, large scale simulation of the outer membrane from Pseudomonas aeruginosa. Using the MARTINI force field approach of 4-to-1 atom mapping, we simulate an asymmetrically constructed bilayer with over 1100 rough lipopolysaccharide (LPS) and 3100 16:0-18:1-phosphatidylethanolamine. We achieve 90-fold improvement in computational efficiency on a system much larger than reasonable for all-atom simulation. We also compare a coarse-grained LPS/LPS bilayer simulation with known parameters determined from neutron diffraction.

α-Tocopherol Is Well Designed to Protect Polyunsaturated Phospholipids: MD Simulations.

The presumptive function for alpha-tocopherol (αtoc) in membranes is to protect polyunsaturated lipids against oxidation. Although the chemistry of the process is well established, the role played by molecular structure that we address here with atomistic molecular-dynamics simulations remains controversial. The simulations were run in the constant particle NPT ensemble on hydrated lipid bilayers composed of SDPC (1-stearoyl-2-docosahexaenoylphosphatidylcholine, 18:0-22:6PC) and SOPC (1-stearoyl-2-oleoylphosphatidylcholine, 18:0-18:1PC) in the presence of 20 mol % αtoc at 37°C. SDPC with SA (stearic acid) for the sn-1 chain and DHA (docosahexaenoic acid) for the sn-2 chain is representative of polyunsaturated phospholipids, while SOPC with OA (oleic acid) substituted for the sn-2 chain serves as a monounsaturated control. Solid-state (2)H nuclear magnetic resonance and neutron diffraction experiments provide validation. The simulations demonstrate that high disorder enhances the probability that DHA chains at the sn-2 position in SDPC rise up to the bilayer surface, whereby they encounter the chromanol group on αtoc molecules. This behavior is reflected in the van der Waals energy of interaction between αtoc and acyl chains, and illustrated by density maps of distribution for acyl chains around αtoc molecules that were constructed. An ability to more easily penetrate deep into the bilayer is another attribute conferred upon the chromanol group in αtoc by the high disorder possessed by DHA. By examining the trajectory of single molecules, we found that αtoc flip-flops across the SDPC bilayer on a submicrosecond timescale that is an order-of-magnitude greater than in SOPC. Our results reveal mechanisms by which the sacrificial hydroxyl group on the chromanol group can trap lipid peroxyl radicals within the interior and near the surface of a polyunsaturated membrane. At the same time, water-soluble reducing agents that regenerate αtoc can access the chromanol group when it locates at the surface.

Macrocyclic Oligoesters Incorporating a Cyclotetrasiloxane Ring.

Macrocyclic oligoester structures based on a cyclotetrasiloxane core consisting of tricyclic (60+ atoms) and pentacycylic (130+ atoms) species were identified as the major components of a lipase-mediated transesterification reaction. Moderately hydrophobic solvents with log P values in the range of 2-3 were more suitable than those at lower or higher log P values. Temperature had little effect on total conversion and yield of the oligoester macrocycles, except when a reaction temperature of 100 °C was employed. At this temperature, the amount of the smaller macrocycle was greatly increased, but at the expense of the larger oligoester. For immobilized lipase B from Candida antarctica (N435), longer chain length esters and diols were more conducive to the synthesis of the macrocycles. Langmuir isotherms indicated that monolayers subjected to multiple compression/expansion cycles exhibited a reversible collapse mechanism different from that expected for linear polysiloxanes.

Granular chains with soft boundaries: Slowing the transition to quasiequilibrium.

We present here a detailed numerical study of the dynamical behavior of "soft" uncompressed grains in a granular chain where the grains interact via the intrinsically nonlinear Hertz force. It is well known that such a chain supports the formation of solitary waves (SWs). Here, however, the system response to the material properties of the grains and boundaries is explored further. In particular, we examine the details of the transition of the system from a SW phase to an equilibrium-like (or quasiequilibrium) phase, and for this reason we ignore the effects of dissipation in this study. We find that the soft walls slow the reflection of SWs at the boundaries of the system, which in turn slows the journey to quasiequilibrium. Moreover, the increased grain-wall compression as the boundaries are softened results in fewer average grain-grain contacts at any given time in the quasiequilibrium phase. These effects lead to increased kinetic energy fluctuations in the short term in softer systems. We conclude with a toy model that exploits the results of soft-wall systems. This toy model supports the formation of breather-like entities and may therefore be useful for localizing energy in desired places in the granular chain.

Neutron Scattering at the Intersection of Heart Health Science and Biophysics.

There is an urgent quest for improved heart health. Here, we review how neutron radiation can provide insight into the molecular basis of heart health. Lower cholesterol, a daily intake of aspirin and supplemental vitamin E are argued to all improve heart health. However, the mechanisms behind these common regimens, and others, are not entirely understood. It is not clear why a daily intake of aspirin can help some people with heart disease, and the benefits of vitamin E in the treatment of reperfusion injury have been heavily debated. The molecular impact of cholesterol in the body is still a hot topic. Neutron scattering experiments present a unique opportunity for biophysicists attempting to address these problems. We review some recently published studies that are advancing our understanding of how cholesterol, vitamin E and aspirin work at the molecular level, by studying the impact of these molecules on the cell membrane. These insights engage the broader health science community with new ways of thinking about these molecules.

α-Tocopherol's Location in Membranes Is Not Affected by Their Composition.

To this day, α-tocopherol's (aToc) role in humans is not well known. In previous studies, we have tried to connect aToc's biological function with its location in a lipid bilayer. In the present study, we have determined, by means of small-angle neutron diffraction, that not only is aToc's hydroxyl group located high in the membrane but its tail also resides far from the center of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. In addition, we located aToc's hydroxyl group above the lipid backbone in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and sphingomyelin bilayers, suggesting that aToc's location near the lipid-water interface may be a universal property of vitamin E. In light of these data, how aToc efficiently terminates lipid hydroperoxy radicals at the membrane center remains an open question.

Molecular structures of fluid phosphatidylethanolamine bilayers obtained from simulation-to-experiment comparisons and experimental scattering density profiles.

Following our previous efforts in determining the structures of commonly used PC, PG, and PS bilayers, we continue our studies of fully hydrated, fluid phase PE bilayers. The newly designed parsing scheme for PE bilayers was based on extensive MD simulations, and is utilized in the SDP analysis of both X-ray and neutron (contrast varied) scattering measurements. Obtained experimental scattering form factors are directly compared to our simulation results, and can serve as a benchmark for future developed force fields. Among the evaluated structural parameters, namely, area per lipid A, overall bilayer thickness DB, and hydrocarbon region thickness 2DC, the PE bilayer response to changing temperature is similar to previously studied bilayers with different headgroups. On the other hand, the reduced hydration of PE headgroups, as well as the strong hydrogen bonding between PE headgroups, dramatically affects lateral packing within the bilayer. Despite sharing the same glycerol backbone, a markedly smaller area per lipid distinguishes PE from other bilayers (i.e., PC, PG, and PS) studied to date. Overall, our data are consistent with the notion that lipid headgroups govern bilayer packing, while hydrocarbon chains dominate the bilayer's response to temperature changes.

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes.

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes. We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir-Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.

Small molecule interaction with lipid bilayers: a molecular dynamics study of chlorhexidine.

Chlorhexidine (CHX) is an effective anti-bacterial agent whose mode of action is thought to be the disruption of the cell membrane. We tested the capability of the Slipids all atom force fields using data from neutron scattering and NMR experiments on the drug chlorhexidine in a 1,2-dimyrisoyl-3-sn-phosphatidylcholine (DMPC) membrane. Since it is not known what the charge of the CHX molecule is inside an apolar environment, a neutral, as well as a +1 and +2 charge model for the molecule were created and tested at several concentrations. This study shows that the location of CHX is minorly dependent on concentration, and dominantly reliant on the charge. The effect of adding CHX to DMPC is a thinning of the membrane, thus increasing the area per lipid.

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.

The Observation of Highly Ordered Domains in Membranes with Cholesterol.

Rafts, or functional domains, are transient nano- or mesoscopic structures in the exoplasmic leaflet of the plasma membrane, and are thought to be essential for many cellular processes. Using neutron diffraction and computer modelling, we present evidence for the existence of highly ordered lipid domains in the cholesterol-rich (32.5 mol%) liquid-ordered ([Formula: see text]) phase of dipalmitoylphosphatidylcholine membranes. The liquid ordered phase in one-component lipid membranes has previously been thought to be a homogeneous phase. The presence of highly ordered lipid domains embedded in a disordered lipid matrix implies non-uniform distribution of cholesterol between the two phases. The experimental results are in excellent agreement with recent computer simulations of DPPC/cholesterol complexes [Meinhardt, Vink and Schmid (2013). Proc Natl Acad Sci USA 110(12): 4476-4481], which reported the existence of nanometer size [Formula: see text] domains in a liquid disordered lipid environment.

Tocopherol activity correlates with its location in a membrane: a new perspective on the antioxidant vitamin E.

We show evidence of an antioxidant mechanism for vitamin E which correlates strongly with its physical location in a model lipid bilayer. These data address the overlooked problem of the physical distance between the vitamin's reducing hydrogen and lipid acyl chain radicals. Our combined data from neutron diffraction, NMR, and UV spectroscopy experiments all suggest that reduction of reactive oxygen species and lipid radicals occurs specifically at the membrane's hydrophobic-hydrophilic interface. The latter is possible when the acyl chain "snorkels" to the interface from the hydrocarbon matrix. Moreover, not all model lipids are equal in this regard, as indicated by the small differences in vitamin's location. The present result is a clear example of the importance of lipid diversity in controlling the dynamic structural properties of biological membranes. Importantly, our results suggest that measurements of aToc oxidation kinetics, and its products, should be revisited by taking into consideration the physical properties of the membrane in which the vitamin resides.