Hydrophobic-hydrophilic Interface Research Articles

Biophysical Properties of 2-Arachidonoylglycerol in a Lipid Matrix and its Binding to Recombinant Cannabinoid Receptor CB2

The endogenous cannabinoid ligand, 2-arachidonoylglycerol (2-AG), is a lipid-derived signaling molecule that activates cannabinoid receptors in brain and peripheral tissues. Because of high lipophilicity, 2-AG readily partitions into lipid bilayers. Molecular behavior of 2-AG in bilayers and mechanism of approach to the receptor binding-pocket remain unsolved; approach via lateral diffusion in lipid matrix was suggested according to microsecond-long molecular dynamics simulations. We studied location, structure, dynamics, and kinetics of acyl-chain migration (2-AG→1-AG) of 2-AG in lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by solid-state NMR and neutron diffraction. Binding of 2-AG to purified CB2 receptor functionally reconstituted into liposomes was probed by 2H magic-angle-spinning NMR of headgroup-deuterated 2-AG (2-AG-d5). The exponential-decay constant of 2-AG due to acyl-chain migration at physiological temperature was determined to be 43 min, while molecular characterization of 2-AG was achieved by investigating the temperature-dependence. 1H cross-relaxation rates measured in two-dimensional NOESY revealed that the double-bond region of the arachidonoyl chain (20:4) of 2-AG locates with high probabilities at the upper methylene segments of POPC, with a significant distribution around glycerol. The data agrees with neutron diffraction, exhibiting highest probabilities of those double bonds near the hydrophobic-hydrophilic interface. The arachidonoyl chain shows high flexibility in lipid bilayers. In the presence of CB2, 2H resonances of 2-AG-d5 shifted upfield with line-broadening. The upfield shift was absent when the pocket was blocked with a high affinity agonist like CP-55,940, demonstrating specificity of 2-AG interaction with the CB2 binding-pocket. The induced shift suggests a model of rapid exchange of 2-AG between free and CB2-bound states; discussions regarding other possibilities will be presented. The enrichment of 2-AG in lipid bilayers and the conformational flexibility of 2-AG are likely to be critical for an efficient interaction with CB2.

Critical Stretching and Pores in Bolalipid Membrane from Flexible String Model

Phase diagram of a bolalipid membrane for critical stretching and pore formation is derived analytically using microscopic flexible strings model [1,2] of monopolar lipid and bolalipid [3] membranes. We use free energy density functional E of the laterally stretched membrane with pore radius R: E = E_0 + (π⊥2R⊥4/2N)∂⊥2Ft(a)/∂a⊥2 + Pπ⊥2R⊥4/Na − PπR⊥2+2ΓπR under the applied stretching pressure P and area per lipid a of the self-assembled membrane of N lipids before stretching, and pore linear tension Γ. The hydrophobic part of the lipid chain's free energy Ft was calculated previously for monopolar [1] and bolalipid [2] membranes using self-consistent microscopic model with entropic repulsion between flexible lipid chains. Finding a from the free energy minimum condition: ∂Ft/∂a+γa=0 , with surface tension γ at the hydrophobic-hydrophilic interface, and substituting it into ∂2Ft(a)/∂a2 we find analytic expression for the coefficient in front of R⊥4 in the elastic energy. Using E we derive phase diagrams for bolalipid and monopolar lipid membranes and compare their stability, i.e. critical pressures Pc of pore formation. An enhancement of Pc of the bolalipid membrane relative to monopolar lipid bilayer is a consequence of relative suppression of the midmembrane entropy of the membranespanning bolalipid chains with respect to the lipid tails entropy in bilayer midplane region [2]. Consequences of this effect for stability against pores formation of the bolalipid membrane of the archaea cell is investigated.1. Mukhin, S.I., Baoukina, S., Analytical derivation of thermodynamic characteristics of lipid bilayer from a flexible string model. Phys. Rev. E 71, 061918 (2005).2. Mukhin, S.I., Kheyfets, B.B., Analytical approach to thermodynamics of bolalipid membranes. Phys. Rev. E 82, 051901 (2010).3. Bulacu, M., Periole, X., Marrink, S.J., In Silico Design of Robust Bolalipid Membranes. Biomacromolecules 13, 196 (2011).

Structure, Folding Dynamics, and Amyloidogenesis of D76N β2-Microglobulin: ROLES OF SHEAR FLOW, HYDROPHOBIC SURFACES, AND α-CRYSTALLIN

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.

Solvent-Induced α- to 310-Helix Transition of an Amphiphilic Peptide

The amphiphilic peptide of the triacylglycerol lipase derived from Pseudomonas aeruginosa plays a critical role in guarding the gate for ligand access. Conformations of this peptide at several water-oil interfaces and in protein environments were compared using atomistic simulations with explicit solvents. In oil-containing solvents, this peptide is able to retain a folded structure. Interestingly, when the peptide is immersed in a low-polarity solvent environment, it exhibits a "coalesced" helix structure, which has both α- and 3(10)-helix components. The observation that the 3(10)-helical conformation is populated in a highly nonpolar environment is consistent with a previous report on polymethylalanine. Frequent interconversions of the secondary structure (between α-helix and 3(10)-helix) of the peptide are also observed. We further studied how this solvent-induced structural transition may be connected to the trigger mechanism of lipase gating and how the lipase senses the hydrophobic-hydrophilic interface.

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.

Enzyme and acid catalyzed degradation of PEG45-b-PBO0,6,9-b-PCL60 micelles: Increased hydrolytic stability by engineering the hydrophilic–hydrophobic interface

Acid and Pseudomonas (P.) cepacia lipase catalyzed ester hydrolysis were evaluated for block copolymer micelles generated from low polydispersity PEG45-b-PBOn-b-PCL60 (n = 0, 6, 9). Moving the hydrophilic–hydrophobic junction away from the PCL micelle core–water interface by inserting a short hydrophobic non-hydrolyzable PBO segment between the PEG and PCL blocks was studied as a strategy for tuning the micelle hydrolytic stability. 1H NMR was applied in evaluating the micelle and solution compositions and to determine kinetic parameters. Acid and lipase catalyzed micelle hydrolysis proceed by distinctly different routes. Micelles from the triblock copolymers PEG45-b-PBOn-b-PCL60 (n = 6, 9) are observed to react substantially slower and persist intact longer in the presence of both strong acids and lipase enzymes than micelles of the parent diblock copolymer (PEG45-b-PCL60).

Study of surfactant alcohols with various chemical moieties at the hydrophilic–hydrophobic interface

The melting behavior, the solubility, and the influence of hydrogen bonds were analyzed for a series of single- and double-tailed surfactant alcohols. Various effects such as the presence of free amides or the intermolecular spacing were found to be important factors for increasing or decreasing the melting temperature of a surfactant. Furthermore, we present a model for the packing of diamido-lipids and study the temperature-dependence of the IR signals.

Single Molecule Force Spectroscopy Studies on the Hydrophobic Hydration

Hydrophobic interactions underpin many biological self-assembly processes, including protein folding and assembly, ligand-receptor interaction, and micelle and membrane formation. Recent theoretic work by Lum, Chandler and Weeks highlighted the distinct mechanisms for the hydrophobic hydration of small solutes and larger ones. Small solutes can be incorporated into surrounding water molecules by simple entropy driven fluctuations of water, whereas solvation of larger solutes requires the formation of hydrophobic-hydrophilic interface and is an enthalpy-dominated process. However, experimentally verifying this theory is fraught with difficulties. Here, using atomic force microscopy (AFM) based single-molecule force spectroscopy, we quantitatively determine the contribution of entropy and enthalpy for the hydrophobic hydration of polystyrene nanospheres. Our work is based upon pioneering work done by Li and Walker. However, we present a novel data analysis strategy to obtain the free energy profile for the hydrophobic hydration process from the force-extension curves in a model-free fashion. The entropy and enthalpy for the hydrophobic hydration of the polystyrene sphere are in good agreement with theoretic predictions. We focus on the hydration of the nanospheres instead of the stretched polystyrene chains, which allows direct measurement of the crossover length for the two distinct hydrophobic hydration mechanisms of hydration of small and large particles. Our experimental results show that the crossover length is ∼ 1 nm and can be tuned by the solvent conditions. Thus, our experiments directly support the length-dependent hydrophobic hydration theory and provide a novel way for the quantification of the free energy for the hydrophobic hydration. Since hydrophobic interactions occur at different length scales in biological systems, we anticipate that our results will be helpful for the understanding of the distinct effect of hydrophobic interactions in various self-assembly process.

An automated two-phase system for hydrogel microbead production

Polymeric beads have been used for protection and delivery of bioactive materials, such as drugs and cells, for different biomedical applications. Here, we present a generic two-phase system for the production of polymeric microbeads of gellan gum or alginate, based on a combination of in situ polymerization and phase separation. Polymer droplets, dispensed using a syringe pump, formed polymeric microbeads while passing through a hydrophobic phase. These were then crosslinked, and thus stabilized, in a hydrophilic phase as they crossed through the hydrophobic–hydrophilic interface. The system can be adapted to different applications by replacing the bioactive material and the hydrophobic and/or the hydrophilic phases. The size of the microbeads was dependent on the system parameters, such as needle size and solution flow rate. The size and morphology of the microbeads produced by the proposed system were uniform, when parameters were kept constant. This system was successfully used for generating polymeric microbeads with encapsulated fluorescent beads, cell suspensions and cell aggregates proving its ability for generating bioactive carriers that can potentially be used for drug delivery and cell therapy.

Effect of the addition of alkanols of different topology to dipalmitoyl-phosphatidylcholine vesicles in the presence of gramicidin

In the present work, we analyze the effect of incorporation of the nonanol family (e.g., 1-Nonanol (1-N), 5-Nonanol (5-N), and 2,6-Dimethyl-4-Heptanol (2,6-DH)) into DPPC LUVs in the presence of different gramicidin concentrations. The principal aim of this work is to study the effect of alkanols solubilization on the physicochemical properties of lipid bilayers in the presence of peptide trans-membrane channels, that is, the effects of nonanol family in the interface of lipid–peptide region, considering that the study provides the analysis of a ternary system by direct excitation as well as by Fluorescence Resonance Energy Transfer. Fluorescence measurements were carried out at 20°C after direct excitation of the extrinsic probe or by Fluorescence Resonance Energy Transfer (FRET) from the tryptophan group of gramicidin. Alkanol incorporation decreases with increasing gramicidin content and branching of the additives. 1-N generates most important changes in the inner part of the bilayer, where it produces an increase in bulk acyl chain mobility. Similarly, 1-N significantly modifies the properties of the hydrophilic–hydrophobic interface region sensed by Laurdan, increasing the polarity of the probe microenvironment and/or increasing the relaxation time of interfacial water molecules. On the other hand, 1-N produces a decrease in PDA fluorescence lifetime, a result that can be explained by a significant amount of water entrance to the inner part of the bilayer. The same behavior was observed when pseudo-first-order quenching rate constants by oxygen were measured. 1-N produces an increase in mobility/solubility of the oxygen in the lipid membrane, an effect that is more noticeable in the deep region of the bilayer sensed by PDA, in the absence and in the presence of 2mol% of Gr. 1-N incorporation produces a greater reduction in GP value than 5-N and 2,6-DH when Laurdan was excited by FRET. These results show that 1-N has the greatest effect in the lipidic domains near the gramicidin channel. On the other hand, excimer–monomer ratios of PDA obtained by FRET show that 1-N reduces the lateral mobility of acyl chains near the lipid–gramicidin interface when gramicidin concentration in the lipid bilayer increases. This effect is more noticeable than that obtained by direct irradiation of the probe in the presence of 5-N and 2,6-DH. On the other hand, the addition of the three alkanols in the presence of Gr produces a noticeable increase in the water permeability, particularly for 1-N. In this context, we propose a scheme that represents the effect of 1-nonanol on the water outflow in DPPC LUVs in the absence and in the presence of Gr.

Differential Dynamic and Structural Behavior of Lipid-Cholesterol Domains in Model Membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).

Membrane-Perturbing Properties of Two Arg-Rich Paddle Domains from Voltage-Gated Sensors in the KvAP and HsapBK K+ Channels

Voltage-gated K(+) channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a "paddle" domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the K(v) channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic-hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

The Conformation of Amyloidogenic Peptides is Governed by (Transient) Interfaces and Influenced by Metal Ion Complexation

Amyloid formation of peptides is related to neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. Amyloid fibrils form after conversion of soluble peptides to structures rich in beta-sheets. Recently, the early stages of beta-sheet formation and amyloid oligomers are considered to be involved in neurotoxicity. However, conditions for the onset of the process are poorly understood.Our strategy to elucidate the molecular mechanisms of beta-sheet formation involves tailor-made model peptides with defined secondary structure propensities and binding sites for metal ions. Their reactions to various triggers are studied separately and in combination. The central focus is on the binding to interfaces. Various surface-sensitive infrared and X-ray methods combined with a Langmuir film balance setup are applied.We have shown that amyloid formation in 2D is governed by the hydrophobic-hydrophilic interface and it is not comparable to the behavior of amyloidogenic peptides in bulk. Depending on the stability of an alpha-helical intermediate, the interface can enhance or inhibit beta-sheet formation. The strongest trigger for beta-sheet formation was identified to be the parallel arrangement of intermediates. Minor effects on the peptide conformation are exerted by metal ion complexation or lipid layers. The effect of metal ion complexation depends on a delicate balance of complex stabilities and conformational energies.In conclusion, our study implies that transient air-water interfaces occurring during any in vitro experiment may lead to amyloid seeding. This partly explains why many studies on amyloid formation are controversial and poorly reproducible regarding kinetics.

Triggers for β-Sheet Formation at the Hydrophobic–Hydrophilic Interface: High Concentration, In-Plane Orientational Order, and Metal Ion Complexation

Amyloid formation plays a causative role in neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Soluble peptides form β-sheets that subsequently rearrange into fibrils and deposit as amyloid plaques. Many parameters trigger and influence the onset of the β-sheet formation. Early stages are recently discussed to be cell-toxic. Aiming at understanding various triggers such as interactions with hydrophobic-hydrophilic interfaces and metal ion complexation and their interplay, we investigated a set of model peptides at the air-water interface. We are using a general approach to a variety of diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes that are connected to amyloid formation. Surface sensitive techniques combined with film balance measurements have been used to assess the conformation of the peptides and their orientation at the air-water interface (IR reflection-absorption spectroscopy). Additionally, the structures of the peptide layers were characterized by grazing incidence X-ray diffraction and X-ray reflectivity. The peptides adsorb to the air-water interface and immediately adopt an α-helical conformation. This helical intermediate transforms into β-sheets upon further triggering. The factors that result in β-sheet formation are dependent on the peptide sequence. In general, the interface has the strongest effect on peptide conformation compared to high concentrations or metal ions. Metal ions are able to prevent aggregation in bulk but not at the interface. At the interface, metal ion complexation has only minor effects on the peptide secondary structure, influencing the in-plane structure that is formed in two dimensions. At the air-water interface, increased concentrations or a parallel arrangement of the α-helical intermediates are the most effective triggers. This study reveals the role of various triggers for β-sheet formation and their complex interplay. Our main finding is that the hydrophobic-hydrophilic interface largely governs the conformation of peptides. Therefore, the present study implies that special care is needed when interpreting data that may be affected by different amounts or types of interfaces during experimentation.

Theory of Domain Formation on Model Myelin Monolayer System

Lipid domains indicate the coexistence of thermodynamic phases of lipid molecules at hydrophobic-hydrophilic interface. Domain size, shape and distribution are directly affected by the line tension (λ) and dipole density difference (m) at domain boundaries [1-2]. In this work, a thermodynamic equation expressing domain size distribution was derived and fitted to domain distributions from fluorescence images of both healthy (control) and experimental allergic encephalomyelitis (EAE) cytoplasmic (CYT) model myelin monolayers. From these fits we simultaneously extract λ (in units of fN) and m (in units of pC/m2). Both parameters λ and m decrease with increasing surface pressure (Π). Moreover, the control monolayer had higher values of λ and m compare with EAE monolayer for all values of Π where domains can be observed clearly. Based on the difference of these values, EAE monolayer seems to be more ‘stressed’ than control monolayer. This indicates an important relationship between Π, domain size and distribution, λ and m, which can be used to point out the potential cause of demyelinating diseases like Multiple Sclerosis (MS) at molecular scale.

Inter-Domain Line Tension Induced by Hydrophobic Lipid Tails in a Lipid Membrane

We consider domain of liquid condensed (LC) lipids surrounded by liquid expanded (LE) phase in a membrane. We use flexible strings model of lipid chains [1]. A greater/smaller flexural rigidity is ascribed to LC / LE lipids respectively. Fluctuating single chain statistically swipes a cylindrical volume. In a mono-component bilayer these cylinders form a close pack. But at the LC/LE boundary the cylinders of the two different diameters can’t form a close pack. Hence, an area per lipid is greater at the boundary than in the bulk of domains. In our model this excessive area leads to a local decrease within the inter-domain boundary of the surface tension at the hydrophilic-hydrophobic interface. If the LC and LE lipids differ only by the flexural rigidity of hydrocarbon chains, then the (flat) boundary energy is negative, Fig. 1, with consequences for domains size distribution.[1] S.I. Mukhin and S.V. Baoukina, Phys. Rev. E71, 061918 (2005). Fig. 1: A) Calculated LC/LE boundary line tension vs flexural rigidities ratio, A- and B- mark LC and LE lipids respectively; B) calculated relative surface tensions difference in the bulk (v) and at domain boundary (b).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Creating Surface Properties Using a Palette of Hydrophobins.

Small secreted proteins called hydrophobins play diverse roles in the life cycle of filamentous fungi. For example, the hydrophobin SC3 of Schizophyllum commune is involved in aerial hyphae formation, cell-wall assembly and attachment to hydrophobic surfaces. Hydrophobins are capable of self-assembly at a hydrophilic-hydrophobic interface, resulting in the formation of an amphipathic film. This amphipathic film can make hydrophobic surfaces of a liquid or a solid material wettable, while a hydrophilic surface can be turned into a hydrophobic one. These properties, among others, make hydrophobins of interest for medical and technical applications. For instance, hydrophobins can be used to purify proteins from complex mixtures; to reduce the friction of materials; to increase the biocompatibility of medical implants; to increase the solubility of water insoluble drugs; and to immobilize enzymes, for example, biosensor surfaces.

Designed Fluorescent Probes Reveal Interactions between Amyloid-β(1–40) Peptides and GM1 Gangliosides in Micelles and Lipid Vesicles

A hallmark of the common Alzheimer's disease (AD) is the pathological conversion of its amphiphatic amyloid-β (Aβ) peptide into neurotoxic aggregates. In AD patients, these aggregates are often found to be tightly associated with neuronal GM1 ganglioside lipids, suggesting an involvement of GM1 not only in aggregate formation but also in neurotoxic events. Significant interactions were found between micelles made of newly synthesized fluorescent GM1 gangliosides labeled in the polar headgroup or the hydrophobic chain and Aβ(1–40) peptide labeled with a BODIPY-FL-C1 fluorophore at positions 12 and 26, respectively. From an analysis of energy transfer between the different fluorescence labels and their location in the molecules, we were able to place the Aβ peptide inside GM1 micelles, close to the hydrophobic-hydrophilic interface. Large unilamellar vesicles composed of a raftlike GM1/bSM/cholesterol lipid composition doped with labeled GM1 at various positions also interact with labeled Aβ peptide tagged to amino acids 2 or 26. A faster energy transfer was observed from the Aβ peptide to bilayers doped with 581/591-BODIPY-C11-GM1 in the nonpolar part of the lipid compared with 581/591-BODIPY-C5-GM1 residing in the polar headgroup. These data are compatible with a clustering process of GM1 molecules, an effect that not only increases the Aβ peptide affinity, but also causes a pronounced Aβ peptide penetration deeper into the lipid membrane; all these factors are potentially involved in Aβ peptide aggregate formation due to an altered ganglioside metabolism found in AD patients.

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 microg ml(-1)) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (beta(1-3),beta(1-6)-glucan) and beta(1-3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.

Annular Protofibrils Are a Structurally and Functionally Distinct Type of Amyloid Oligomer

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (alphaAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in alphaAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric alpha-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a beta barrel structural motif.