Hannah Dies

The Observation of Highly Ordered Domains in Membranes with Cholesterol.

Rafts, or functional domains, are transient nano- or mesoscopic structures in the exoplasmic leaflet of the plasma membrane, and are thought to be essential for many cellular processes. Using neutron diffraction and computer modelling, we present evidence for the existence of highly ordered lipid domains in the cholesterol-rich (32.5 mol%) liquid-ordered () phase of dipalmitoylphosphatidylcholine membranes. The liquid ordered phase in one-component lipid membranes has previously been thought to be a homogeneous phase. The presence of highly ordered lipid domains embedded in a disordered lipid matrix implies non-uniform distribution of cholesterol between the two phases. The experimental results are in excellent agreement with recent computer simulations of DPPC/cholesterol complexes [Meinhardt, Vink and Schmid (2013). Proc Natl Acad Sci USA 110(12): 4476–4481], which reported the existence of nanometer size domains in a liquid disordered lipid environment.

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.

The organization of melatonin in lipid membranes

Melatonin is a hormone that has been shown to have protective effects in several diseases that are associated with cholesterol dysregulation, including cardiovascular disease, Alzheimers disease, and certain types of cancers. We studied the interaction of melatonin with model membranes made of dimyristoylphosphatidylcholine (DMPC) at melatonin concentrations ranging from 0.5mol% to 30mol%. From 2-dimensional X-ray diffraction measurements, we find that melatonin induces a re-ordering of the lipid membrane that is strongly dependent on the melatonin concentration. At low melatonin concentrations, we observe the presence of melatonin-enriched patches in the membrane, which are significantly thinner than the lipid bilayer. The melatonin molecules were found to align parallel to the lipid tails in these patches. At high melatonin concentrations of 30mol%, we observe a highly ordered melatonin structure that is uniform throughout the membrane, where the melatonin molecules align parallel to the bilayers and one melatonin molecule associates with 2 lipid molecules. Understanding the organization and interactions of melatonin in membranes, and how these are dependent on the concentration, may shed light into its anti-amyloidogenic, antioxidative and photoprotective properties and help develop a structural basis for these properties.

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.

The Interaction of Bio-Molecules with Lipid Membranes Studied by X-ray Diffraction

Abstract For the past 100 years, X-ray diffraction has been a powerful and indispensable tool to study the structure of matter. The challenge when studying molecular ordering in biological materials is their inherent disorder and strong fluctuations, which often suppress the formation of Bragg peaks. In the case of membranes, X-rays can detect molecules inside and confined between membranes. In this article we review examples to highlight the capabilities and accomplishments of X-ray scattering for the determination of membrane structure. X-ray diffraction gives quantitative information about partitioning of a small molecule, ethanol, in lipid bilayers. By taking amyloid-β peptides as examples, it is demonstrated that the position of peptides in lipid membranes can be determined with high precision. Confinement between membranes can organize molecules, as is the case for a mono-nucleotide, adenosine monophosphate, and the resulting pattern might be important to understand the formation of short RNA strands. With new approaches and techniques, and the increasingly powerful and capable devices, X-ray diffraction will continue to be the work horse for the determination of molecular structure in biological materials.

Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol’s condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content.