Revealing cholesterol effects on PEGylated HSPC liposomes using AF4-MALS and simultaneous small- and wide-angle X-ray scattering.

Liposome development is of great interest owing to increasing requirements for efficient drug carriers. The structural features and thermal stability of such liposomes are crucial in drug transport and delivery. Reported here are the results of the structural characterization of PEGylated liposomes via small- and wide-angle X-ray scattering and an asymmetric flow field-flow fractionation (AF4) system coupled with differential refractive-index detection, multi-angle light scattering (MALS) and dynamic light scattering. This integrated analysis of the exemplar PEGylated liposome formed from hydrogenated soy phosphatid-yl-choline (HSPC) with the addition of cholesterol reveals an average hydro-dynamic radius (R h) of 52 nm with 10% polydispersity, a comparable radius of gyration (R g) and a major liposome particle mass of 118 kDa. The local bilayer structure of the liposome is found to have asymmetric electronic density profiles in the inner and outer leaflets, sandwiched by two PEGylated outer layers ca 5 nm thick. Cholesterol was found to effectively intervene in lipid chain packing, resulting in the thickening of the liposome bilayer, an increase in the area per lipid and an increase in liposome size, especially in the fluid phase of the liposome. These cholesterol effects show signs of saturation at cholesterol concentrations above ca 1:5 cholesterol:lipid molar ratio.

Cholesterol twists the transmembrane Di-Gly region of amyloid-precursor protein.

Nearly 95% of Alzheimer's disease (AD) occurs sporadically without genetic linkage. Aging, hypertension, high cholesterol content, and diabetes are known nongenomic risk factors of AD. Aggregation of Aß peptides is an initial event of AD pathogenesis. Aß peptides are catabolic products of a type I membrane protein called amyloid precursor protein (APP). Aß40 is the major product, whereas the 2-residue-longer version, Aß42, induces amyloid plaque formation in the AD brain. Since cholesterol content is one risk factor for sporadic AD, we aimed to explore whether cholesterol in the membrane affects the structure of the APP transmembrane region, thereby modulating the γ-secretase cutting behavior. Here, we synthesized several peptides containing the APP transmembrane region (sequence 693-726, corresponding to the Aß22-55 sequence) with one or two Cys mutations for spin labeling. We performed three electron spin resonance experiments to examine the structural changes of the peptides in liposomes composed of dioleoyl phosphatidylcholine and different cholesterol content. Our results show that cholesterol increases membrane thickness by 10% and peptide length accordingly. We identified that the di-glycine region of Aß36-40 (sequence VGGVV) exhibits the most profound change in response to cholesterol compared with other segments, explaining how the presence of cholesterol affects the γ-secretase cutting site. This study provides spectroscopic evidence showing how cholesterol modulates the structure of the APP transmembrane region in a lipid bilayer.

Conformational Changes of α-Crystallin Proteins Induced by Heat Stress.

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.

Aging and Charge Compensation Effects of the Rechargeable Aqueous Zinc/Copper Hexacyanoferrate Battery Elucidated Using In Situ X-ray Techniques.

The zinc/copper hexacyanoferrate (Zn/CuHCF) cell has gained attention as an aqueous rechargeable zinc-ion battery (ZIB) owing to its open framework, excellent rate capability, and high safety. However, both the Zn anode and the CuHCF cathode show unavoidable signs of aging during cycling, though the underlying mechanisms have remained somewhat ambiguous. Here, we present an in-depth study of the CuHCF cathode by employing various X-ray spectroscopic techniques. This allows us to distinguish between structure-related aging effects and charge compensation processes associated with electroactive metal centers upon Zn2+ ion insertion/deinsertion. By combining high-angle annular dark-field-scanning electron transmission microscopy, X-ray absorption spectroscopy (XAS), X-ray photoelectron spectroscopy, and elemental analysis, we reconstruct the picture of both the bulk and the surface. First, we identify a set of previously debated X-ray diffraction peaks appearing at early stages of cycling (below 200 cycles) in CuHCF. Our data suggest that these peaks are unrelated to hypothetical ZnxCu1-xHCF phases or to oxidic phases, but are caused by partial intercalation of ZnSO4 into graphitic carbon. We further conclude that Cu is the unstable species during aging, whose dissolution is significant at the surface of the CuHCF particles. This triggers Zn2+ ions to enter newly formed Cu vacancies, in addition to native Fe vacancies already present in the bulk, which causes a reduction of nearby metal sites. This is distinct from the charge compensation process where both the Cu2+/Cu+ and Fe3+/Fe2+ redox couples participate throughout the bulk. By tracking the K-edge fluorescence using operando XAS coupled with cyclic voltammetry, we successfully link the aging effect to the activation of the Fe3+/Fe2+ redox couple as a consequence of Cu dissolution. This explains the progressive increase in the voltage of the charge/discharge plateaus upon repeated cycling. We also find that SO42- anions reversibly insert into CuHCF during charge. Our work clarifies several intriguing structural and redox-mediated aging mechanisms in the CuHCF cathode and pinpoints parameters that correlate with the performance, which will hold importance for the development of future Prussian blue analogue-type cathodes for aqueous rechargeable ZIBs.

Probing Electrochemical Potential Differences over the Solid/Liquid Interface in Li-Ion Battery Model Systems.

The electrochemical potential difference (ΔµÌ…) is the driving force for the transfer of a charged species from one phase to another in a redox reaction. In Li-ion batteries (LIBs), ΔµÌ… values for both electrons and Li-ions play an important role in the charge-transfer kinetics at the electrode/electrolyte interfaces. Because of the lack of suitable measurement techniques, little is known about how ΔµÌ… affects the redox reactions occurring at the solid/liquid interfaces during LIB operation. Herein, we outline the relations between different potentials and show how ambient pressure photoelectron spectroscopy (APPES) can be used to follow changes in ΔµÌ…e over the solid/liquid interfaces operando by measuring the kinetic energy (KE) shifts of the electrolyte core levels. The KE shift versus applied voltage shows a linear dependence of ∼1 eV/V during charging of the electrical double layer and during solid electrolyte interphase formation. This agrees with the expected results for an ideally polarizable interface. During lithiation, the slope changes drastically. We propose a model to explain this based on charge transfer over the solid/liquid interface.

Rhombohedral trap for studying molecular oligomerization in membranes: application to daptomycin.

A persistent problem in the studies of membrane-active peptides, including antimicrobial peptides and pathogenic amyloidal peptides, is the lack of methods for investigating their molecular configurations in membranes. These peptides spontaneously bind to membranes from solutions, and often form oligomers that induce changes of membrane permeability. For antimicrobials, such actions appear to relate to the antimicrobial mechanisms, but for amyloidal peptides, the oligomerization has been linked to neurodegenerative diseases. In many cases, no further understanding of such oligomerization has been achieved due to the lack of structural information. In this article, we will demonstrate a method of trapping such peptide oligomers in a rhombohedral (R) phase of lipid so that the oligomers can be subjected to 3D diffraction analysis. The conditions for forming the R phase and the electron density distribution in the rhombohedral unit cell provide information about peptide-lipid interactions and the molecular size of the trapped oligomer. Such information cannot be obtained from membranes in the planar configuration. For illustration, we apply this method to daptomycin, an FDA-approved antibiotic that attacks membranes containing phosphatidylglycerol, in the presence of calcium ions. We have successfully used the brominated phosphatidylglycerol to perform bromine-atom anomalous diffraction in the rhombohedral phase containing daptomycin and calcium ions. The preliminary results apparently exhibit diffraction data related to daptomycin oligomers. We believe that this method will also be applicable to the difficult problems related to amyloidal peptides, such as amyloid beta of Alzheimer's disease.

The opposing effect of butanol and butyric acid on the abundance of bromide and iodide at the aqueous solution-air interface.

The efficient oxidation of iodide and bromide at the aqueous solution-air interface of the ocean or of sea spray aerosol particles had been suggested to be related to their surface propensity. The ubiquitous presence of organic material at the ocean surface calls for an assessment of the impact of often surface-active organic compounds on the interfacial density of halide ions. We used in situ X-ray photoelectron spectroscopy with a liquid micro-jet to obtain chemical composition information at aqueous solution-vapor interfaces from mixed aqueous solutions containing bromide or iodide and 1-butanol or butyric acid as organic surfactants. Core level spectra of Br 3d, Na 2s, C 1s and O 1s at ca. 160 eV kinetic energy and core level spectra of I 4d and O 1s at ca. 400 eV kinetic energy are compared for solutions with 1-butanol and butyric acid as a function of organic concentration. A simple model was developed to account for the attenuation of photoelectrons by the aliphatic carbon layer of the surfactants and for changing local density of bromide and iodide in response to the presence of the surfactants. We observed that 1-butanol increases the interfacial density of bromide by 25%, while butyric acid reduces it by 40%, both in comparison to the pure aqueous halide solution. Qualitatively similar behavior was observed for the case of iodide. Classical molecular dynamics simulations failed to reproduce the details of the response of the halide ions to the presence of the two organics. This is attributed to the lack of correct monovalent ion parameters at low concentration possibly leading to an overestimation of the halide ion concentration at the interface in absence of organics. The results clearly demonstrate that organic surfactants change the electrostatic interactions near the interface with headgroup specific effects. This has implications for halogen activation processes specifically when oxidants interact with halide ions at the aqueous solution-air interfaces of the ocean surface or sea spray aerosol particles.

Interplay of entropy and enthalpy in peptide binding to zwitterionic phospholipid membranes as revealed from membrane thinning.

Membrane thinning that resulted from peptide-binding is observed via temperature dependent small-angle X-ray scattering (SAXS). The result reveals a mean thermal thinning rate of 0.038 Å K-1 for the neat unilamellar vesicles (ULVs) of a zwitterionic phospholipid of 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (diC20:1PC) in the temperature range of 285-312 K. The thinning effect promotes greatly the association between a model antimicrobial peptide melittin and the ULV. Scaling the observed isothermal melittin-ULV bilayer thinning to that measured using low-angle X-ray diffraction from the melittin-multilamellar membranes of defined peptide-to-lipid ratios establishes temperature-dependent binding isotherms χb of the peptide-ULV as a function of free peptide concentration in solution. From the binding isotherms, temperature-dependent peptide-membrane binding constant K(T) is extracted on the basis of a modified Gouy-Chapman model. Changes in K(T) follow the linearized van't Hoff equation ln K(T) ∝ -ΔHT-1 with a constant enthalpy change ΔH = 9.6 kcal mol-1, suggesting an entropy-driven binding process prior to membrane pore formation. Correspondingly, a five-fold enhancement of K is observed in the temperature range studied. The peptide-binding strength is found to follow the growth trend of the membrane thermal thinning rate better than the lipid chain length of the three phosphocholine-based ULVs of diCn:1PC with n = 18, 20, and 22.

Comparison of the Effects of Daptomycin on Bacterial and Model Membranes.

Daptomycin is a phosphatidylglycerol specific, calcium-dependent membrane-active antibiotic that has been approved for the treatment of Gram-positive infections. A recent Bacillus subtilis study found that daptomycin clustered into fluid lipid domains of bacterial membranes and the membrane binding was correlated with dislocation of peripheral membrane proteins and depolarization of membrane potential. In particular, the study disproved the existence of daptomycin ion channels. Our purpose here is to study how daptomycin interacts with lipid bilayers to understand the observed phenomena on bacterial membranes. We performed new types of experiments using aspirated giant vesicles with an ion leakage indicator, making comparisons between daptomycin and ionomycin, performing vesicle-vesicle transfers, and measuring daptomycin binding to fluid phase versus gel phase bilayers and bilayers including cholesterol. Our findings are entirely consistent with the observations for bacterial membranes. In addition, daptomycin is found to cause ion leakage through the membrane only if its concentration in the membrane is over a certain threshold. The ion leakage caused by daptomycin is transient. It occurs only when daptomycin binds the membrane for the first time; afterward, they cease to induce ion leakage. The ion leakage effect of daptomycin cannot be transferred from one membrane to another. The level of membrane binding of daptomycin is reduced in the gel phase versus the fluid phase. Cholesterol also weakens the membrane binding of daptomycin. The combination of membrane concentration threshold and differential binding is significant. This could be a reason why daptomycin discriminates between eukaryotic and prokaryotic cell membranes.

The Penetration Depth for Hanatoxin Partitioning into the Membrane Hydrocarbon Core Measured with Neutron Reflectivity.

Hanatoxin (HaTx) from spider venom works as an inhibitor of Kv2.1 channels, most likely by interacting with the voltage sensor (VS). However, the way in which this water-soluble peptide modifies the gating remains poorly understood as the VS is deeply embedded within the bilayer, although it would change its position depending on the membrane potential. To determine whether HaTx can indeed bind to the VS, the depth at which HaTx penetrates into the POPC membranes was measured with neutron reflectivity. Our results successfully demonstrate that HaTx penetrates into the membrane hydrocarbon core (∼9 Šfrom the membrane surface), not lying on the membrane-water interface as reported for another voltage sensor toxin (VSTx). This difference in penetration depth suggests that the two toxins fix the voltage sensors at different positions with respect to the membrane normal, thereby explaining their different inhibitory effects on the channels. In particular, results from MD simulations constrained by our penetration data clearly demonstrate an appropriate orientation for HaTx to interact with the membranes, which is in line with the biochemical information derived from stopped-flow analysis through delineation of the toxin-VS binding interface.

X-ray Reflectivity Studies on the Mixed Langmuir-Blodgett Monolayers of Thiol-Capped Gold Nanoparticles, Dipalmitoylphosphatidylcholine, and Sodium Dodecyl Sulfate.

Langmuir-Blodgett monolayers of thiolated gold nanoparticles mixed with dipalmitoylphosphatidylcholine/sodium dodecyl sulfate (DPPC/SDS) were investigated by combining the X-ray reflectivity, grazing-incident scattering, and TEM analyses to reveal the in-depth and in-plane organization and the 2D morphology of such mixed monolayers. It was found that the addition of a charged single-tail surfactant to the thiolated Au nanoparticle monolayer helps to stabilize the Au nanoparticle monolayer and to strengthen the mechanical property of the mixed monolayer film. For mixing with lipids, it was found that the thiolated gold nanoparticles could be pushed on top of the lipid monolayer when the mixed monolayer is compressed. At a typical comparable total surface area ratio of gold nanoparticle to lipid, the thiolated gold nanoparticles could form a uniform domain on top of the DPPC monolayer. When there are more thiolated gold nanoparticles than that could be supported by the lipid monolayer, domain overlapping could occur to form bilayer gold nanoparticle domains at some regions. At low total surface area ratio of thiolated gold nanoparticle to lipid, the thiolated gold nanoparticles tend to form a connected threadlike aggregation structure. Evidently, the morphology of the thiolated gold nanoparticle monolayer is highly depending on the total surface area ratio of the thiolated gold nanoparticle to lipid. SDS is found to have a dispersion power capable of dispersing the originally uniform Au-8C nanoparticle domain of the mixed Au-8C/DPPC monolayer into a foamlike structure for the mixed Au-8C/SDS/DPPC monolayer. It is evident that not only the concentration ratio but also the size and shape of the template formed by the amphiphilic molecules and their interaction with the thiolated gold nanoparticles can all have great effects on the organizational structure as well as morphology of the thiolated gold nanoparticle monolayer.

A surface-stabilized ozonide triggers bromide oxidation at the aqueous solution-vapour interface.

Oxidation of bromide in aqueous environments initiates the formation of molecular halogen compounds, which is important for the global tropospheric ozone budget. In the aqueous bulk, oxidation of bromide by ozone involves a [Br•OOO-] complex as intermediate. Here we report liquid jet X-ray photoelectron spectroscopy measurements that provide direct experimental evidence for the ozonide and establish its propensity for the solution-vapour interface. Theoretical calculations support these findings, showing that water stabilizes the ozonide and lowers the energy of the transition state at neutral pH. Kinetic experiments confirm the dominance of the heterogeneous oxidation route established by this precursor at low, atmospherically relevant ozone concentrations. Taken together, our results provide a strong case of different reaction kinetics and mechanisms of reactions occurring at the aqueous phase-vapour interface compared with the bulk aqueous phase.Heterogeneous oxidation of bromide in atmospheric aqueous environments has long been suspected to be accelerated at the interface between aqueous solution and air. Here, the authors provide spectroscopic, kinetic and theoretical evidence for a rate limiting, surface active ozonide formed at the interface.

Molecular State of the Membrane-Active Antibiotic Daptomycin.

Membrane-active antibiotics are potential alternatives to the resistance-prone conventional antibiotics. Daptomycin, a cyclic lipopeptide, is the only membrane-active antibiotic approved by the U.S. Food and Drug Administration so far. The drug interacts with the cytoplasmic membranes of Gram-positive pathogens, causing membrane permeabilization to ions and cell death. The antibiotic activity is calcium-ion dependent and correlates with the target membrane's content of phosphatidylglycerol (PG). For such a complex reaction with membranes, it has been difficult to uncover the molecular process that underlies its antibacterial activity. The role of the cofactor, calcium ions, has been confusing. Many have proposed that calcium ions binding to daptomycin is a precondition for membrane interaction. Here, we report our findings on the molecular state of daptomycin before and after its membrane-binding reaction, particularly at therapeutic concentrations in the low micromolar range. We were able to perform small-angle x-ray scattering at sufficiently low daptomycin concentrations to determine that the molecules are monomeric before membrane binding. By careful circular dichroism (CD) analyses of daptomycin with Ca2+ and PG-containing membranes, we found that there are only two states identifiable by CD, one before and another after membrane binding; all other CD spectra are linear combinations of the two. Before membrane binding, the molecular state of daptomycin as defined by CD is the same with or without calcium ions. We are able to determine the stoichiometric ratios of the membrane-binding reaction. The stoichiometric ratio of daptomycin to calcium is 2:3. The stoichiometric ratio of daptomycin to PG is ∼1:1 if only the PG lipids in the outer leaflets of membranes are accessible to daptomycin.

Measurement of Hanatoxin-Induced Membrane Thinning with Lamellar X-ray Diffraction.

Membrane perturbation induced by cysteine-rich peptides is a crucial biological phenomenon but scarcely investigated, in particular with effective biophysical-chemical methodologies. Hanatoxin (HaTx), a 35-residue polypeptide from spider venom, works as an inhibitor of drk1 (Kv2.1) channels, most likely by interacting with the voltage-sensor. However, how this water-soluble peptide modifies the gating remains poorly understood, as the voltage sensor was proposed to be deeply embedded within the bilayer. To see how HaTx interacts with phospholipid bilayers, we observe the toxin-induced perturbation on POPC/DOPG-membranes through measurements of the change in membrane thickness. Lamellar X-ray diffraction (LXD) was applied on stacked planar bilayers in the near-fully hydrated state. The results provide quantitative evidence for the membrane thinning in a concentration-dependent manner, leading to novel and direct combinatory approaches by discovering how to investigate such a biologically relevant interaction between gating-modifier toxins and phospholipid bilayers.

Hanatoxin inserts into phospholipid membranes without pore formation.

Hanatoxin (HaTx), a 35-residue polypeptide from spider venom, functions as an inhibitor of Kv2.1 channels by interacting with phospholipids prior to affecting the voltage-sensor. However, how this water-soluble peptide modifies the gating remains poorly understood, as the voltage-sensor is deeply embedded within the bilayer. To determine how HaTx interacts with phospholipid bilayers, in this study, we examined the toxin-induced partitioning of liposomal membranes. HPLC-results from high-speed spin-down vesicles with HaTx demonstrated direct binding. Dynamic light scattering (DLS) and leakage assay results further indicated that neither membrane pores nor membrane fragmentations were observed in the presence of HaTx. To clarify the binding details, Langmuir trough experiments were performed with phospholipid monolayers by mimicking the external leaflet of membrane bilayers, indicating the involvement of acyl chains in such interactions between HaTx and phospholipids. Our current study thus describes the interaction pattern of HaTx with vesicle membranes, defining a membrane-partitioning mechanism for peptide insertion involving the membrane hydrocarbon core without pore formation.

Chemical Composition and Properties of the Liquid-Vapor Interface of Aqueous C1 to C4 Monofunctional Acid and Alcohol Solutions.

The liquid-vapor interface is playing an important role in aerosol and cloud chemistry in cloud droplet activation by aerosol particles and potentially also in ice nucleation. We have employed the surface sensitive and chemically selective X-ray photoelectron spectroscopy (XPS) technique to examine the liquid-vapor interface for mixtures of water and small alcohols or small carboxylic acids (C1 to C4), abundant chemicals in the atmosphere in concentration ranges relevant for cloud chemistry or aerosol particles at the point of activation into a cloud droplet. A linear correlation was found between the headgroup carbon 1s core-level signal intensity and the surface excess derived from literature surface tension data with the offset being explained by the bulk contribution to the photoemission signal. The relative interfacial enhancement of the carboxylic acids over the carboxylates at the same bulk concentration was found to be highest (nearly 20) for propionic acid/propionate and still about 5 for formic acid/formate, also in fair agreement with surface tension measurements. This provides direct spectroscopic evidence for high carboxylic acid concentrations at aqueous solution-air interfaces that may be responsible for acid catalyzed chemistry under moderately acidic conditions with respect to their bulk aqueous phase acidity constant. By assessing the ratio of aliphatic to headgroup C 1s signal intensities XPS also provides information about the orientation of the molecules. The results indicate an increasing orientation of alcohols and neutral acids toward the surface normal as a function of chain length, along with increasing importance of lateral hydrophobic interactions at higher surface coverage. In turn, the carboxylate ions exhibit stronger orientation toward the surface normal than the corresponding neutral acids, likely caused by the stronger hydration of the charged headgroup.

Influence of Fe substitution on the Jahn-Teller distortion and orbital anisotropy in orthorhombic Y(Mn1-xFex)O3 epitaxial films.

Multiferroic YMn1-xFexO3(020) (x = 0.125, 0.25, 0.50) epitaxial thin films with an orthorhombic structure (space group Pbnm) were prepared on a YAlO3(010) substrate by pulsed-laser deposition. Upon Fe substitution, the b-axis was clearly shortened, whereas the a- and c-axes were slightly lengthened based on XRD analysis. To understand the influence of orbital polarization and the Jahn-Teller effect of Mn(3+) on Fe substitution and also the local octahedral-site distortion of Fe(3+) in an environment of Jahn-Teller-active Mn(3+) ions in YMn1-xFexO3 films, we measured the polarization-dependent X-ray absorption spectra at the Mn-L2,3 and Fe-L2,3 edges, and also simulated the experimental spectra using configuration-interaction multiplet calculations. Although Δeg for the Mn(3+) ion decreased from 0.9 eV in pure YMnO3 to 0.6 eV in the half-Fe-substituted sample, a single eg electron was still strongly constrained to the d3y(2)-r(2) orbital for all the Fe concentrations tested. The largest Δeg, 0.5 eV, for the Fe(3+) ion was derived for a sample with 12.5% Fe substitution, and gradually decreased to 0.15 eV for the half-Fe-substituted sample. The local octahedral-site distortion of the Fe(3+) ion inside the YMnO3 lattice was similar to that of the Mn(3+) ion, whereas the Jahn-Teller distortion and GdFeO3-type distortion of the Mn(3+) ion were decreased by the spherical high-spin Fe(3+) ions. The combination of the experimental and theoretical data provides both profound insight into the variation of the Jahn-Teller distortion and orbital anisotropy and instructive information about the magnetic structures in these orthorhombic YMn1-xFexO3 thin films.

Comparative Study of the Condensing Effects of Ergosterol and Cholesterol.

Cholesterol, due to its condensing effect, is considered an important regulator of membrane thickness. Other sterols, due to their structural similarities to cholesterol, are often assumed to have a universal effect on membrane properties similar to the condensing effect of cholesterol, albeit possibly to different degrees. We used x-ray diffraction to investigate this assumption. By the combination of lamellar diffraction and grazing-angle scattering, we measured the membrane thickness and the tilt-angle distribution of the lipid's hydrocarbon chains. This method is sensitive to phase separation, which is important for examining the miscibility of sterols and phospholipids. Mixtures of ergosterol or cholesterol with dimyristoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, and dioleoylphosphatidylcholine were systematically studied. We found that mixing ergosterol with phospholipids into a single phase became increasingly difficult with higher sterol concentrations and also with higher concentrations of unsaturated lipid chains. The only condensing effect of ergosterol was found in dimyristoylphosphatidylcholine, although the effect was less than one-third of the effect of cholesterol. Unlike cholesterol, ergosterol could not maintain a fixed electron density profile of the surrounding lipids independent of hydration. In dioleoylphosphatidylcholine and palmitoyloleoylphosphatidylcholine, ergosterol made the membranes thinner, opposite to the effect of cholesterol. In all cases, the tilt-angle variation of the chain diffraction was consistent with the membrane thickness changes measured by lamellar diffraction, i.e., a thickening was always associated with a reduction of chain tilt angles. Our findings do not support the notion that different sterols have a universal behavior that differs only in degree.

Membrane-mediated amyloid formation of PrP 106-126: A kinetic study.

PrP 106-126 conserves the pathogenic and physicochemical properties of the Scrapie isoform of the prion protein. PrP 106-126 and other amyloidal proteins are capable of inducing ion permeability through cell membranes, and this property may represent the common primary mechanism of pathogenesis in the amyloid-related degenerative diseases. However, for many amyloidal proteins, despite numerous phenomenological observations of their interactions with membranes, it has been difficult to determine the molecular mechanisms by which the proteins cause ion permeability. One approach that has not been undertaken is the kinetic study of protein-membrane interactions. We found that the reaction time constant of the interaction between PrP 106-126 and membranes is suitable for such studies. The kinetic experiment with giant lipid vesicles showed that the membrane area first increased by peptide binding but then decreased. The membrane area decrease was coincidental with appearance of extramembranous aggregates including lipid molecules. Sometimes, the membrane area would increase again followed by another decrease. The kinetic experiment with small vesicles was monitored by circular dichroism for peptide conformation changes. The results are consistent with a molecular simulation following a simple set of well-defined rules. We deduced that at the molecular level the formation of peptide amyloids incorporated lipid molecules as part of the aggregates. Most importantly the amyloid aggregates desorbed from the lipid bilayer, consistent with the macroscopic phenomena observed with giant vesicles. Thus we conclude that the main effect of membrane-mediated amyloid formation is extraction of lipid molecules from the membrane. We discuss the likelihood of this effect on membrane ion permeability.