The role of shear forces in primary and secondary nucleation of amyloid fibrils.

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A[Formula: see text]42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.

Wrapping anisotropic microgel particles in lipid membranes: Effects of particle shape and membrane rigidity.

Cellular engulfment and uptake of macromolecular assemblies or nanoparticles via endocytosis can be associated to both healthy and disease-related biological processes as well as delivery of drug nanoparticles and potential nanotoxicity of pollutants. Depending on the physical and chemical properties of the system, the adsorbed particles may remain at the membrane surface, become wrapped by the membrane, or translocate across the membrane through an endocytosis-like process. In this paper, we address the question of how the wrapping of colloidal particles by lipid membranes can be controlled by the shape of the particles, the particle-membrane adhesion energy, the membrane phase behavior, and the membrane-bending rigidity. We use a model system composed of soft core-shell microgel particles with spherical and ellipsoidal shapes, together with phospholipid membranes with varying composition. Confocal microscopy data clearly demonstrate how tuning of these basic properties of particles and membranes can be used to direct wrapping and membrane deformation and the organization of the particles at the membrane. The deep-wrapped states are more favorable for ellipsoidal than for spherical microgel particles of similar volume. Theoretical calculations for fixed adhesion strength predict the opposite behavior-wrapping becomes more difficult with increasing aspect ratio. The comparison with the experiments implies that the microgel adhesion strength must increase with increasing particle stretching. Considering the versatility offered by microgels systems to be synthesized with different shapes, functionalizations, and mechanical properties, the present findings further inspire future studies involving nanoparticle-membrane interactions relevant for the design of novel biomaterials and therapeutic applications.

Retardation of Aß42 fibril formation by apolipoprotein A-I and recombinant HDL particles.

The double nucleation mechanism of amyloid ß (Aß) peptide aggregation is retained from buffer to cerebrospinal fluid (CSF) but with reduced rate of all microscopic processes. Here, we used a bottom-up approach to identify retarding factors in CSF. We investigated the Aß42 fibril formation as a function of time in the absence and presence of apolipoprotein A-I (ApoA-I), recombinant high-density lipoprotein (rHDL) particles, or lipid vesicles. A retardation was observed in the presence of ApoA-I or rHDL particles, most pronounced with ApoA-I, but not with lipid vesicles. Global kinetic analysis implies that rHDL interferes with secondary nucleation. The effect of ApoA-I could best be described as an interference with secondary and to a smaller extent primary nucleation. Using surface plasmon resonance and microfluidics diffusional sizing analyses, we find that both rHDL and ApoA-I interact with Aß42 fibrils but not Aß42 monomer, thus the effect on kinetics seems to involve interference with the catalytic surface for secondary nucleation. The Aß42 fibrils were imaged using cryogenic-electron microscopy and found to be longer when formed in the presence of ApoA-I or rHDL, compared to formation in buffer. A retarding effect, as observed in CSF, could be replicated using a simpler system, from key components present in CSF but purified from a CSF-free host. However, the effect of CSF is stronger implying the presence of additional retarding factors.

Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering.

The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid ß, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of α synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-α-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils.

Solubility of Foreign Molecules in Stratum Corneum Brick and Mortar Structure.

The barrier function of the skin is mainly assured by its outermost layer, stratum corneum (SC). One key aspect in predicting dermal drug delivery and in safety assessment of skin exposure to chemicals is the need to determine the amount of chemical that is taken up into the SC. We here present a strategy that allows for direct measures of the amount of various solid chemicals that can be dissolved in the SC in any environmental relative humidity (RH). A main advantage of the presented method is that it distinguishes between molecules that are dissolved within the SC and molecules that are not dissolved but might be present at, for example, the skin surface. In addition, the method allows for studies of uptake of hydrophobic chemicals without the need to use organic solvents. The strategy relies on the differences in the molecular properties of the added molecules in the dissolved and the excess states, employing detection methods that act as a dynamic filter to spot only one of the fractions, either the dissolved molecules or the excess solid molecules. By measuring the solubility in SC and delipidized SC at the same RHs, the same method can be used to estimate the distribution of the added chemical between the extracellular lipids and corneocytes at different hydration conditions. The solubility in porcine SC is shown to vary with hydration, which has implications for the molecular uptake and transport across the skin. The findings highlight the importance of assessing the chemical uptake at hydration conditions relevant to the specific applications. The methodology presented in this study can also be generalized to study the solubility and partitioning of chemicals in other heterogeneous materials with complex composition and structure.

Molecular Mobility in Keratin-Rich Materials Monitored by Nuclear Magnetic Resonance: A Tool for the Evaluation of Structure-Giving Properties.

Keratins are structural proteins that are abundant in human skin, nails, and hair, where they provide mechanical strength. In the present study, we investigate the molecular mobilities and structures of three keratin-rich materials with clearly different mechanical properties: nails, stratum corneum (upper layer of epidermis), and keratinocytes (from lower layer of epidermis). We use solid-state NMR on natural-abundance 13C to characterize small changes in molecular dynamics in these biological materials with close to atomistic resolution. One strong advantage of this method is that it detects small fractions of mobile components in a molecularly complex material while it simultaneously gives information on the rigid components in the very same sample. The molecular mobility can be linked to mechanical material properties in different conditions, including hydration or exposure to osmolytes or organic solvents. Importantly, the study revealed that the response to both hydration and addition of urea is clearly different for the nail keratin compared to the stratum corneum keratin. The comparative examination of these materials may provide a better understanding of skin diseases originating from keratin malfunction and contributes to the design and development of new materials.

α-Synuclein Interaction with Lipid Bilayer Discs.

α-Synuclein (aSyn) is a 140 residue long protein present in presynaptic termini of nerve cells. The protein is associated with Parkinson's disease, in which case it has been found to self-assemble into long amyloid fibrils forming intracellular inclusions that are also rich in lipids. Furthermore, its synaptic function is proposed to involve interaction with lipid membranes, and hence, it is of interest to understand aSyn-lipid membrane interactions in detail. In this paper we report on the interaction of aSyn with model membranes in the form of lipid bilayer discs. Using a combination of cryogenic transmission electron microscopy and small-angle neutron scattering, we show that circular discs undergo a significant shape transition after the adsorption of aSyn. When aSyn self-assembles into fibrils, aSyn molecules desorb from the bilayer discs, allowing them to recover to their original shape. Interestingly, the desorption process has an all-or-none character, resulting in a binary coexistence of circular bilayer discs with no adsorbed aSyn and deformed bilayer discs having a maximum amount of adsorbed protein. The observed coexistence is consistent with the recent finding of cooperative aSyn adsorption to anionic lipid bilayers.

Molecular dynamics simulations and solid-state nuclear magnetic resonance spectroscopy measurements of C-H bond order parameters and effective correlation times in a POPC-GM3 bilayer.

Glycolipids such as gangliosides affect the properties of lipid membranes and in extension the interactions between membranes and other biomolecules like proteins. To better understand how the properties of individual lipid molecules can contribute to shape the functional aspects of a membrane, the spatial restriction and dynamics of C-H bond segments can be measured using nuclear magnetic resonance (NMR) spectroscopy. We combine solid-state NMR spectroscopy with all-atom molecular dynamics (MD) simulations to investigate how ganglioside GM3 affects the bilayer structure and dynamics of C-H bond segments. These two methods yield reorientational correlation functions, molecular profiles of C-H bond order parameters |SCH| and effective correlation times τe, which we compare for lipids in POPC bilayers with and without 30 mol% GM3. Our results revealed that all C-H segments of POPC reorient slower in the presence of GM3 and that the defining features of the GM3-POPC bilayer lie in the GM3 headgroup; it gives the bilayer an extended headgroup layer with high order (|SCH| up to 0.3-0.4) and slow dynamics (τe up to 100 ns), a character that may be mechanistically important in ganglioside interactions with other biomolecules.

Assembling responsive microgels at responsive lipid membranes.

Directed colloidal self-assembly at fluid interfaces can have a large impact in the fields of nanotechnology, materials, and biomedical sciences. The ability to control interfacial self-assembly relies on the fine interplay between bulk and surface interactions. Here, we investigate the interfacial assembly of thermoresponsive microgels and lipogels at the surface of giant unilamellar vesicles (GUVs) consisting of phospholipids bilayers with different compositions. By altering the properties of the lipid membrane and the microgel particles, it is possible to control the adsorption/desorption processes as well as the organization and dynamics of the colloids at the vesicle surface. No translocation of the microgels and lipogels through the membrane was observed for any of the membrane compositions and temperatures investigated. The lipid membranes with fluid chains provide highly dynamic interfaces that can host and mediate long-range ordering into 2D hexagonal crystals. This is in clear contrast to the conditions when the membranes are composed of lipids with solid chains, where there is no crystalline arrangement, and most of the particles desorb from the membrane. Likewise, we show that in segregated membranes, the soft microgel colloids form closely packed 2D crystals on the fluid bilayer domains, while hardly any particles adhere to the more solid bilayer domains. These findings thus present an approach for selective and controlled colloidal assembly at lipid membranes, opening routes toward the development of tunable soft materials.

On the Aggregation of Apolipoprotein A-I.

In vivo, apolipoprotein A-I (ApoA-I) is commonly found together with lipids in so-called lipoprotein particles. The protein has also been associated with several diseases-such as atherosclerosis and amyloidosis-where insoluble aggregates containing ApoA-I are deposited in various organs or arteries. The deposited ApoA-I has been found in the form of amyloid fibrils, suggesting that amyloid formation may be involved in the development of these diseases. In the present study we investigated ApoA-I aggregation into amyloid fibrils and other aggregate morphologies. We studied the aggregation of wildtype ApoA-I as well as a disease-associated mutant, ApoA-I K107Δ, under different solution conditions. The aggregation was followed using thioflavin T fluorescence intensity. For selected samples the aggregates formed were characterized in terms of size, secondary structure content, and morphology using circular dichroism spectroscopy, dynamic light scattering, atomic force microscopy and cryo transmission electron microscopy. We find that ApoA-I may form globular protein-only condensates, in which the α-helical conformation of the protein is retained. The protein in its unmodified form appears resistant to amyloid formation; however, the conversion into amyloid fibrils rich in ß-sheet is facilitated by oxidation or mutation. In particular, the K107Δ mutant shows higher amyloid formation propensity, and the end state appears to be a co-existence of ß-sheet rich amyloid fibrils and α-helix-rich condensates.

Strong inhibition of peptide amyloid formation by a fatty acid.

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer's and Parkinson's disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68-78 of the protein α-synuclein involved in Parkinson's disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.

Purification and HDL-like particle formation of apolipoprotein A-I after co-expression with the EDDIE mutant of Npro autoprotease.

Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in E. coli. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus intact. ApoA-I was then purified using a five-step procedure composed of anion exchange chromatography, immobilized metal ion affinity chromatography, hydrophobic interaction chromatography, boiling and size exclusion chromatography. This led to protein of high purity as confirmed with SDS-PAGE and mass spectrometry. The purified ApoA-I formed discoidal objects in the presence of zwitterionic phospholipid DMPC, showing its retained function of interacting with lipids. The protocol was also tested by expression and purification of two ApoA-I mutants, both of which could be purified in the same manner as the wildtype, showing the robustness of the protocol.

Solid and fluid segments within the same molecule of stratum corneum ceramide lipid.

The outer layer of the skin, stratum corneum (SC) is an efficient transport barrier and it tolerates mechanical deformation. At physiological conditions, the majority of SC lipids are solid, while the presence of a small amount of fluid lipids is considered crucial for SC barrier and material properties. Here we use solid-state and diffusion nuclear magnetic resonance to characterize the composition and molecular dynamics of the fluid lipid fraction in SC model lipids, focusing on the role of the essential SC lipid CER EOS, which is a ceramide esterified omega-hydroxy sphingosine linoleate with very long chain. We show that both rigid and mobile structures are present within the same CER EOS molecule, and that the linoleate segments undergo fast isotropic reorientation while exhibiting extraordinarily slow self-diffusion. The characterization of this unusual self-assembly in SC lipids provides deepened insight into the molecular arrangement in the SC extracellular lipid matrix and the role of CER EOS linoleate in the healthy and diseased skin.

The plant dehydrin Lti30 stabilizes lipid lamellar structures in varying hydration conditions.

A major challenge to plant growth and survival are changes in temperature and diminishing water supply. During acute temperature and water stress, plants often express stress proteins, such as dehydrins, which are intrinsically disordered hydrophilic proteins. In this article, we investigated how the dehydrin Lti30 from Arabidopsis thaliana stabilizes membrane systems that are exposed to large changes in hydration. We also compared the effects of Lti30 on membranes with those of the simple osmolytes urea and trimethylamine N-oxide. Using X-ray diffraction and solid-state NMR, we studied lipid-protein self-assembly at varying hydration levels. We made the following observations: 1) the association of Lti30 with anionic membranes relies on electrostatic attraction, and the protein is located in the bilayer interfacial membrane region; 2) Lti30 can stabilize the lamellar multilayer structure, making it insensitive to variations in water content; 3) in lipid systems with a composition similar to those present in some seeds and plants, dehydrin can prevent the formation of nonlamellar phases upon drying, which may be crucial for maintaining membrane integrity; and 4) Lti30 stabilizes bilayer structures both at high and low water contents, whereas the small osmolyte molecules mainly prevent dehydration-induced transitions. These results corroborate the idea that dehydrins are part of a sensitive and multifaceted regulatory mechanism that protects plant cells against stress.

Quantification of the amount of mobile components in intact stratum corneum with natural-abundance 13C solid-state NMR.

The outermost layer of the skin is the stratum corneum (SC), which is mainly comprised of solid proteins and lipids. Minor amounts of mobile proteins and lipids are crucial for the macroscopic properties of the SC, including softness, elasticity and barrier function. Still this minor number of mobile components are not well characterized in terms of structure or amount. Conventional quantitative direct polarization (Q-DP) 13C solid-state NMR gives signal amplitudes proportional to concentrations, but fails to quantify the SC mobile components because of spectral overlap with the overwhelming signals from the solids. Spectral editing with the INEPT scheme suppresses the signals from solids, but also modulates the amplitudes of the mobile components depending on their values of the transverse relaxation times T2, scalar couplings JCH, and number of covalently bound hydrogens nH. This study describes a quantitative INEPT (Q-INEPT) method relying on systematic variation of the INEPT timing variables to estimate T2, JCH, nH, and amplitude for each of the resolved resonances from the mobile components. Q-INEPT is validated with a series of model systems containing molecules with different hydrophobicity and dynamics. For selected systems where Q-DP is applicable, the results of Q-INEPT and Q-DP are similar with respect to the linearity and uncertainty of the obtained molar ratios. Utilizing a reference compound with known concentration, we quantify the concentrations of mobile lipids and proteins within the mainly solid SC. By melting all lipids at high temperature, we obtain the total lipid concentration. These Q-INEPT results are the first steps towards a quantitative understanding of the relations between mobile component concentrations and SC macroscopic properties.

Tracking solvents in the skin through atomically resolved measurements of molecular mobility in intact stratum corneum.

Solvents are commonly used in pharmaceutical and cosmetic formulations and sanitary products and cleansers. The uptake of solvent into the skin may change the molecular organization of skin lipids and proteins, which may in turn alter the protective skin barrier function. We herein examine the molecular effects of 10 different solvents on the outermost layer of skin, the stratum corneum (SC), using polarization transfer solid-state NMR on natural abundance 13C in intact SC. With this approach it is possible to characterize the molecular dynamics of solvent molecules when present inside intact SC and to simultaneously monitor the effects caused by the added solvent on SC lipids and protein components. All solvents investigated cause an increased fluidity of SC lipids, with the most prominent effects shown for the apolar hydrocarbon solvents and 2-propanol. However, no solvent other than water shows the ability to fluidize amino acids in the keratin filaments. The solvent molecules themselves show reduced molecular mobility when incorporated in the SC matrix. Changes in the molecular properties of the SC, and in particular alternation in the balance between solid and fluid SC components, may have significant influences on the macroscopic SC barrier properties as well as mechanical properties of the skin. Deepened understanding of molecular effects of foreign compounds in SC fluidity can therefore have strong impact on the development of skin products in pharmaceutical, cosmetic, and sanitary applications.

Effect of cholesterol on the molecular structure and transitions in a clinical-grade lung surfactant extract.

The lipid-protein film covering the interface of the lung alveolar in mammals is vital for proper lung function and its deficiency is related to a range of diseases. Here we present a molecular-level characterization of a clinical-grade porcine lung surfactant extract using a multitechnique approach consisting of [Formula: see text]-[Formula: see text] solid-state nuclear magnetic spectroscopy, small- and wide-angle X-ray scattering, and mass spectrometry. The detailed characterization presented for reconstituted membranes of a lung extract demonstrates that the molecular structure of lung surfactant strongly depends on the concentration of cholesterol. If cholesterol makes up about 11% of the total dry weight of lung surfactant, the surfactant extract adopts a single liquid-ordered lamellar phase, [Formula: see text], at physiological temperatures. This [Formula: see text] phase gradually changes into a liquid-disordered lamellar phase, [Formula: see text], when the temperature is increased by a few degrees. In the absence of cholesterol the system segregates into one lamellar gel phase and one [Formula: see text] phase. Remarkably, it was possible to measure a large set of order parameter magnitudes [Formula: see text] from the liquid-disordered and -ordered lamellar phases and assign them to specific C-H bonds of the phospholipids in the biological extract with no use of isotopic labeling. These findings with molecular details on lung surfactant mixtures together with the presented NMR methodology may guide further development of pulmonary surfactant pharmaceuticals that better mimic the physiological self-assembly compositions for treatment of pathological states such as respiratory distress syndrome.

Secondary nucleation of monomers on fibril surface dominates α-synuclein aggregation and provides autocatalytic amyloid amplification.

Parkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing α-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.

Influence of polar co-solutes and salt on the hydration of lipid membranes.

The influence of the co-solutes TMAO, urea, and NaCl on the hydration repulsion between lipid membranes is investigated in a combined experimental/simulation approach. Pressure-hydration curves obtained via sorption experiments reveal that the repulsion significantly increases when the membranes are loaded with co-solutes, most strongly for TMAO. As a result, the co-solutes retain additional water molecules and therefore provide membranes with a fluid and more physiological environment. The experimental data are quantitatively reproduced in complementary solvent-explicit atomistic molecular dynamics simulations, which yield the chemical potential of water. Simulation analysis reveals that the additional repulsion arises from the osmotic pressure generated by the co-solutes, an effect which is maximal for TMAO, due to its unfavorable interactions with the lipid headgroup layer and its extraordinarily high osmotic coefficient.

Controlling water evaporation through self-assembly.

Water evaporation concerns all land-living organisms, as ambient air is dryer than their corresponding equilibrium humidity. Contrarily to plants, mammals are covered with a skin that not only hinders evaporation but also maintains its rate at a nearly constant value, independently of air humidity. Here, we show that simple amphiphiles/water systems reproduce this behavior, which suggests a common underlying mechanism originating from responding self-assembly structures. The composition and structure gradients arising from the evaporation process were characterized using optical microscopy, infrared microscopy, and small-angle X-ray scattering. We observed a thin and dry outer phase that responds to changes in air humidity by increasing its thickness as the air becomes dryer, which decreases its permeability to water, thus counterbalancing the increase in the evaporation driving force. This thin and dry outer phase therefore shields the systems from humidity variations. Such a feedback loop achieves a homeostatic regulation of water evaporation.