Laura Toppozini

Structure of Cholesterol in Lipid Rafts.

Rafts, or functional domains, are transient nano-or mesoscopic structures in the plasma membrane and are thought to be essential for many cellular processes such as signal transduction, adhesion, trafficking, and lipid or protein sorting. Observations of these membrane heterogeneities have proven challenging, as they are thought to be both small and short lived. With a combination of coarse-grained molecular dynamics simulations and neutron diffraction using deuterium labeled cholesterol molecules, we observe raftlike structures and determine the ordering of the cholesterol molecules in binary cholesterol-containing lipid membranes. From coarse-grained computer simulations, heterogenous membranes structures were observed and characterized as small, ordered domains. Neutron diffraction was used to study the lateral structure of the cholesterol molecules. We find pairs of strongly bound cholesterol molecules in the liquid-disordered phase, in accordance with the umbrella model. Bragg peaks corresponding to ordering of the cholesterol molecules in the raftlike structures were observed and indexed by two different structures: a monoclinic structure of ordered cholesterol pairs of alternating direction in equilibrium with cholesterol plaques, i.e., triclinic cholesterol bilayers.

The interaction between amyloid-β peptides and anionic lipid membranes containing cholesterol and melatonin.

One of the hallmarks of Alzheimers disease is the formation of senile plaques, primarily consisting of amyloid- (A) peptides. Peptide-membrane and peptide-lipid interactions are thought to be crucial in this process. We studied the interaction of A and A peptides with anionic lipid membranes made of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphoserine (DMPS) using X-ray diffraction. We compare the experimentally determined electron densities in the gel state of the membranes with density calculations from peptide structures reported in the Protein Data Bank in order to determine the position of the peptide in the bilayers. The full length peptide A was found to embed in the hydrocarbon core of the anionic lipid bilayers. Two populations were found for the A peptide: (1) membrane-bound states in the hydrophilic head group region of the bilayers, where the peptides align parallel to the membranes, and (2) an embedded state in the bilayer center. Aging plays an important role in the development of Alzheimers, in particular with respect to changes in cholesterol and melatonin levels in the brain tissue. Immiscible cholesterol plaques were created by addition of 30 mol% cholesterol to the anionic membranes. The A peptides were found to strongly interact with the lipid bilayers, displacing further cholesterol molecules into the plaques, effectively lowering the cholesterol concentration in the membranes and increasing the total fraction of cholesterol plaques. Addition of 30 mol% melatonin molecules to the anionic membranes drastically reduced the population of the membrane-embedded A state. These results present experimental evidence for an interaction between A peptides, melatonin and cholesterol in lipid membranes.

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%.

Adenosine monophosphate forms ordered arrays in multilamellar lipid matrices: insights into assembly of nucleic acid for primitive life.

A fundamental question of biology is how nucleic acids first assembled and then were incorporated into the earliest forms of cellular life 4 billion years ago. The polymerization of nucleotides is a condensation reaction in which phosphodiester bonds are formed. This reaction cannot occur in aqueous solutions, but guided polymerization in an anhydrous lipid environment could promote a non-enzymatic condensation reaction in which oligomers of single stranded nucleic acids are synthesized. We used X-ray scattering to investigate 5′-adenosine monophosphate (AMP) molecules captured in a multilamellar phospholipid matrix composed of dimyristoylphosphatidylcholine. Bragg peaks corresponding to the lateral organization of the confined AMP molecules were observed. Instead of forming a random array, the AMP molecules are highly entangled, with the phosphate and ribose groups in close proximity. This structure may facilitate polymerization of the nucleotides into RNA-like polymers.

Partitioning of ethanol into lipid membranes and its effect on fluidity and permeability as seen by X-ray and neutron scattering

We present a combined neutron and X-ray scattering investigation to study the effect of ethanol on the molecular structure and dynamics of lipid membranes. 1,2-Dimyristoyl-sn-glycero-3-phoshatidylcholine (DMPC) powder hydrated with a 5 wt% ethanol solution (corresponding to 2 mol% of ethanol) was used in this study. From high-resolution X-ray experiments the position and partitioning of the ethanol molecules in the phospholipid bilayers was determined in their gel and fluid phases. We find that the ethanol molecules reside in the head group region of the bilayers, with 1.6 ethanol molecules per lipid molecule in the gel phase and 1.2 ethanol molecules per lipid molecule in the fluid phase. We find evidence for enhanced permeability in both fluid and gel phases of the phospholipid bilayers in the presence of ethanol molecules. Elastic and quasi-elastic neutron scattering data, obtained using a neutron backscattering spectrometer, was used to study slow, nanosecond molecular dynamics on length scales corresponding to lipid diffusion, acyl chain dynamics and solvent dynamics. While the presence of ethanol molecules had no observable effect on these types of dynamics in the fluid (Lα) phase, the membranes appeared to have a higher degree of order in gel (Lβ) and ripple (Pβ′) phases. In particular, lipid diffusion was found to be slower by a factor of two in the more rigid gel phase when ethanol was present.

Co-existence of gel and fluid lipid domains in single-component phospholipid membranes

Lateral nanostructures in membranes, so-called rafts, are believed to strongly influence membrane properties and functions. The experimental observation of rafts has proven difficult as they are thought to be dynamic structures that likely fluctuate on nano- to microsecond time scales. Using neutron diffraction we present direct experimental evidence for the co-existence of gel and fluid lipid domains in a single-component phospholipid membrane made of DPPC as it undergoes its main phase transition. The coherence length of the neutron beam sets a lower limit for the size of structures that can be observed. Neutron coherence lengths between 30 and 242 A used in this study were obtained by varying the incident neutron energy and the resolution of the neutron spectrometer. We observe Bragg peaks corresponding to co-existing nanometer sized structures, both in out-of-plane and in-plane scans, by tuning the neutron coherence length. During the main phase transition, instead of a continuous transition that shows a pseudo-critical behavior, we observe the co-existence of gel and fluid domains.

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.

Incoherent Neutron Spin-Echo Spectroscopy as an Option to Study Long-Range Lipid Diffusion

Diffusion is the fundamental mechanism for lipids and other molecules to move in a membrane. It is an important process to consider in modelling the formation of membrane structures, such as rafts. Lipid diffusion is mainly studied by two different techniques: incoherent neutron scattering and fluorescence microscopy. Both techniques access distinctly different length scales. While neutron scattering measures diffusion over about 3 lipid diameters, microscopic techniques access motions of lipids over micrometer distances. The diffusion constants which are determined by these two methods often differ by about an order of magnitude, with the neutrons usually seeing a faster lipid diffusion. Different theories are used to describe lipid diffusion in the two experiments. In order to close the “gap” between these two techniques, we propose to study lipid diffusion at mesoscopic length scales using a neutron spin-echo (NSE) spectrometer. We have conducted an experiment in highly oriented, solid supported lipid bilayers to prove the feasibility of performing incoherent NSE on biological samples. Lateral lipid diffusion was measured in a fluid phase model membrane system at a length scale of 12 A. Using the high-energy resolution of the NSE technique, we find evidence for two dynamic processes.

Static magnetic fields enhance lipid order in native plant plasma membrane

We studied molecular order in native plant plasma membranes in the presence of static magnetic fields. Plasma membranes of seeds of the tomato plant were purified, extracted, and applied to a silicon substrate in a buffer suspension and their molecular structure was studied using X-ray diffraction. We observed correlation peaks that we assigned to the lipid and protein components of the plasma membrane. Two field strengths were available: B = 0.126 T and B = 0.208 T. While magnetic fields had no observable effect on protein structure, enhanced lipid order was observed, leading to an increase in the gel components and a decrease in the fluid component of the lipids.

Hydration Water Freezing in Single Supported Lipid Bilayers

We present a high-temperature and high-energy resolution neutron scattering investigation of hydration water freezing in single supported lipid bilayers. Single supported lipid bilayers provide a well-defined biological interface to study hydration water dynamics and coupling to membrane degrees of freedom. Nanosecond molecular motions of membrane and hydration water were studied in the temperature range 240 K < T < 290 K in slow heating and cooling cycles using coherent and incoherent elastic neutron scattering on a backscattering spectrometer. Several freezing and melting transitions were observed. From the length scale dependence of the elastic scattering, these transitions could be assigned to freezing and melting of hydration water dynamics, diffusive lipid, and lipid acyl-tail dynamics. Coupling was investigated by comparing the different freezing and melting temperatures. While it is often speculated that membrane and hydration water dynamics are strongly coupled, we find that membrane and hydration water dynamics are at least partially decoupled in single bilayers.