Richard J. Alsop

Interaction of Aspirin (Acetylsalicylic Acid) with Lipid Membranes

We studied the interaction of Aspirin (acetylsalicylic acid) with lipid membranes using x-ray diffraction for bilayers containing up to 50 mol% of aspirin. From 2D x-ray intensity maps that cover large areas of reciprocal space we determined the position of the ASA molecules in the phospholipid bilayers and the molecular arrangement of the molecules in the plane of the membranes. We present direct experimental evidence that ASA molecules participate in saturated lipid bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and preferably reside in the head group region of the membrane. Up to 50 mol% ASA molecules can be dissolved in this type of bilayer before the lateral membrane organization is disturbed and the membranes are found to form an ordered, 2D crystal-like structure. Furthermore, ASA and cholesterol were found to co-exist in saturated lipid bilayers, with the ASA molecules residing in the head group region and the cholesterol molecules participating in the hydrophobic membrane core.

Solubility of cholesterol in lipid membranes and the formation of immiscible cholesterol plaques at high cholesterol concentrations

The molecular in-plane and out-of-plane structure of dimyristoylphosphatidylcholine (DMPC) membranes containing up to 60 mol% of cholesterol was studied using X-ray diffraction. Up to 37.5 mol% cholesterol could be dissolved in the membranes, resulting in a disordered lateral membrane structure. Highly ordered cholesterol structures were observed at cholesterol concentrations of more than 40 mol% cholesterol. These structures were characterized as immiscible cholesterol plaques, i.e., bilayers of cholesterol molecules coexisting with the lipid bilayer. The cholesterol molecules were found to form a monoclinic structure at 40 mol% cholesterol, which transformed into a triclinic arrangement at the highest concentration of 60 mol%. Monoclinic and triclinic structures were found to coexist at cholesterol concentrations between 50 and 55 mol%.

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

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

The Lipid Bilayer Provides a Site for Cortisone Crystallization at High Cortisone Concentrations.

Cortisone is an injected anti-inflammatory drug that can cause painful side effects known as “steroid flares” which are caused by cortisone crystallizing at the injection site. We used molecular dynamics simulations and X-ray diffraction to study the interaction of cortisone with model lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at drug concentrations from 0 mol% to 50 mol%. Cortisone was found to partition in the lipid bilayer and locate in the hydrophilic to hydrophobic interface of the membranes. Cortisone strongly affects the integrity of the membrane, as quantified by a decreased membrane thickness, increased area per lipid, and decreased lipid tail order parameters. At cortisone concentrations of more than 20 mol%, signals from crystallized cortisone were observed. These crystallites are embedded in the bilayers and orient with the membranes. While the cortisone molecules align parallel to the bilayers at low concentrations, they start to penetrate the hydrophobic core at higher concentrations. Trans-membrane crystallites start to nucleate when the membrane thickness has decreased such that cortisone molecules in the different leaflets can find partners from the opposite leaflet resulting in a non-zero density of cortisone molecules in the bilayer center. We suggest that the lipid bilayer provides a site for cortisone crystallization.

The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes

We prepared highly oriented, multi-lamellar stacks of human red blood cell (RBC) membranes applied on silicon wafers. RBC ghosts were prepared by hemolysis and applied onto functionalized silicon chips and annealed into multi-lamellar RBC membranes. High resolution X-ray diffraction was used to determine the molecular structure of the stacked membranes. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. Our results further support current models of RBC membranes as patchy structures and provide unprecedented structural details of the molecular organization in the different domains.

Effect of shampoo, conditioner and permanent waving on the molecular structure of human hair

The hair is a filamentous biomaterial consisting of the cuticle, the cortex and the medulla, all held together by the cell membrane complex. The cortex mostly consists of helical keratin proteins that spiral together to form coiled-coil dimers, intermediate filaments, micro-fibrils and macro-fibrils. We used X-ray diffraction to study hair structure on the molecular level, at length scales between ∼3–90 Å, in hopes of developing a diagnostic method for diseases affecting hair structure allowing for fast and noninvasive screening. However, such an approach can only be successful if common hair treatments do not affect molecular hair structure. We found that a single use of shampoo and conditioner has no effect on packing of keratin molecules, structure of the intermediate filaments or internal lipid composition of the membrane complex. Permanent waving treatments are known to break and reform disulfide linkages in the hair. Single application of a perming product was found to deeply penetrate the hair and reduce the number of keratin coiled-coils and change the structure of the intermediate filaments. Signals related to the coiled-coil structure of the α-keratin molecules at 5 and 9.5 Å were found to be decreased while a signal associated with the organization of the intermediate filaments at 47 Å was significantly elevated in permed hair. Both these observations are related to breaking of the bonds between two coiled-coil keratin dimers.

The Position of Aβ22-40 and Aβ1-42 in Anionic Lipid Membranes Containing Cholesterol

Amyloid-β peptides interact with cell membranes in the human brain and are associated with neurodegenerative diseases, such as Alzheimer’s disease. An emerging explanation of the molecular mechanism, which results in neurodegeneration, places the cause of neurotoxicity of the amyloid-β peptides on their potentially negative interaction with neuronal membranes. It is known that amyloid-β peptides interact with the membrane, modifying the membrane’s structural and dynamic properties. We present a series of X-ray diffraction experiments on anionic model lipid membranes containing various amounts of cholesterol. These experiments provide experimental evidence for an interaction of both the full length amyloid-β1−42 peptide, and the peptide fragment amyloid-β22−40 with anionic bilayer containing cholesterol. The location of the amyloid-β peptides was determined from these experiments, with the full length peptide embedding into the membrane, and the peptide fragment occupying 2 positions—on the membrane surface and embedded into the membrane core.

Design of hydrated porphyrin-phospholipid bilayers with enhanced magnetic resonance contrast

Computer simulations are used to design more hydrated bilayers, formed from amine-modified porphyrin-phospholipids (PoPs). Experiments confirm that the new constructs give rise to bilayers with greater water content. When chelated with manganese, amine-modified PoPs provide improved contrast for magnetic resonance and are safely used for imaging in vivo.

Membrane-Accelerated Amyloid-β Aggregation and Formation of Cross-β Sheets

Amyloid-β aggregates play a causative role in Alzheimer’s disease. These aggregates are a product of the physical environment provided by the basic neuronal membrane, composed of a lipid bilayer. The intrinsic properties of the lipid bilayer allow amyloid-β peptides to nucleate and form well-ordered cross-β sheets within the membrane. Here, we correlate the aggregation of the hydrophobic fragment of the amyloid-β protein, Aβ25-35, with the hydrophobicity, fluidity, and charge density of a lipid bilayer. We summarize recent biophysical studies of model membranes and relate these to the process of aggregation in physiological systems.

Structural Abnormalities in the Hair of a Patient with a Novel Ribosomopathy.

We report the biophysical characterization of hair from a patient with a de novo ribosomopathy. The patient was diagnosed with a mutation on gene RPS23, which codes for a protein which comprises part of the 40S subunit of the ribosome. The patient presents with a number of phenotypes, including hypotonia, autism, extra teeth, elastic skin, and thin/brittle hair. We combined optical microscopy, tensile tests, and X-ray diffraction experiments on hair samples obtained from the scalp of the patient to a multi-scale characterization of the hair from macroscopic to molecular length scales and observe distinct differences in the biophysical properties in the patient’s hair when compared to hair from other family members. While no differences were observed in the coiled-coil structure of the keratin proteins or the structure of the intermediate filaments, the patient’s hair was 22% thinner, while the Young’s modulus remained roughly constant. The X-ray diffraction results give evidence that the amount of lipids in the cell membrane complex is reduced by 20%, which well accounts for the other observations. The pathologies characterized by these techniques may be used to inform the diagnosis of similar de novo mutations in the future.