1,720,966 research outputs found
X-ray structural investigations of fusion intermediates: Lipid model systems and beyond
No full textWe review recent X-ray diffraction studies of fusion intermediate structures in lipid model membranes. We illustrate what kind of information can be derived on the stalk structure as well as on the energetics of stalk formation. The role of different lipids is elucidated by a structural fusion assay, highlighting the significant modulation of fusion energetics by the lipid stoichiometry. Extending beyond the pure lipid models, we also include recent work probing the interaction of lipid bilayers and synaptic vesicles, from a structural point of view. The review closes with an outlook on X-ray imaging of lipid bilayers as a possible new experimental method to study membrane fusion
Stalk structures in lipid bilayer fusion studied by x-ray diffraction
No full textSoftcover, 182 S.: 44,00 €Softcover, 17x24The fusion of two biological membranes is an important step in many processes on the cellular and sub-cellular level. Understanding the involved interplay of different lipid species, a specialized protein machinery and water on length scales of few nanometers poses a significant challenge to current structural biology. Among several complementary approaches, one strategy is to study the structural rearrangements of the lipid matrix. As the initial step, lipid bilayers must be forced into close contact to form a non-bilayer intermediate termed a stalk. This has been the subject of numerous theoretical studies and simulations, but experimental data on stalks are largely lacking. Currently, the only way to obtain structural information at the required sub-nanometer resolution is x-ray diffraction on the recently discovered “stalk phase” formed by certain lipids. We apply this method to elucidate the effect of lipid composition on stalk geometry and the repulsive forces between lipid bilayers prior to stalk formation. An approach based on differential geometry of electron density isosurfaces is introduced to analyze the curvatures and bending energies of the lipid monolayers. For the first time, this connects experiment-based structures of stalks and the associated bending and hydration energies. In addition, this thesis aims to provide a self-contained introduction to the required background in x-ray diffraction on lipid mesophases and electron density reconstruction
Radiation damage studies in cardiac muscle cells and tissue using microfocused X-ray beams: experiment and simulation
No full textSoft materials are easily affected by radiation damage from intense, focused synchrotron beams, often limiting the use of scanning diffraction experiments to radiation-resistant samples. To minimize radiation damage in experiments on soft tissue and thus to improve data quality, radiation damage needs to be studied as a function of the experimental parameters. Here, the impact of radiation damage in scanning X-ray diffraction experiments on hydrated cardiac muscle cells and tissue is investigated. It is shown how the small-angle diffraction signal is affected by radiation damage upon variation of scan parameters and dose. The experimental study was complemented by simulations of dose distributions for microfocused X-ray beams in soft muscle tissue. As a simulation tool, the Monte Carlo software package EGSnrc was used that is widely used in radiation dosimetry research. Simulations also give additional guidance for a more careful planning of dose distribution in tissue
Energetics of stalk intermediates in membrane fusion are controlled by lipid composition
No full textWe have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 +/- 0.5 angstrom for the investigated lipid compositions
Acyl-Chain Correlation in Membrane Fusion Intermediates: X-Ray Diffraction from the Rhombohedral Lipid Phase
No full textAbstractWe have studied the acyl-chain conformation in stalk phases of model membranes by x-ray diffraction from oriented samples. As an equilibrium lipid phase induced by dehydration, the stalk or rhombohedral phase exhibits lipidic passages (stalks) between adjacent bilayers, representing a presumed intermediate state in membrane fusion. From the detailed analysis of the acyl-chain correlation peak, we deduce the structural parameters of the acyl-chain fluid above, at, and below the transition from the lamellar to rhombohedral state, at the molecular level
Effect of PIP2 on Bilayer Structure and Phase Behavior of DOPC: An X-ray Scattering Study
No full textPhosphatidylinositol 4,5-bis-phosphate (PIP2) is an important lipid in regulation of several cellular processes, particularly membrane fusion. We use X-ray diffraction from solid-supported multilamellar 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/PIP2 samples to study changes in bilayer structure and the lyotropic phase behavior induced by physiologically relevant concentrations of PIP2. Electron-density profiles reconstructed from X-ray reflectivity measurements indicate that PIP2 strongly affects structural parameters such as lipid head-group width, bilayer thickness, and lamellar repeat spacing of DOPC bilayer stacks. In addition, at lower degrees of hydration, a few molar per cent of PIP2 facilitates stalk-phase formation and also leads to formation of a hexagonal phase, which is not observed in pure DOPC. These results indicate that the role of PIP2 in membrane fusion could be, in part, due to its effect on the properties of the lipid bilayer matrix. Furthermore, coexistence of two lamellar phases with different lattice constants is observed in single-component PIP2 samples
Stalk formation as a function of lipid composition studied by X-ray reflectivity
No full textAbstractWe have investigated the structure and interaction of solid-supported multilamellar phospholipid bilayers in view of stalk formation as model systems for membrane fusion. The multi-component bilayers were composed of ternary and quaternary mixtures, containing phosphatidylcholines, phosphatidylethanolamines, sphingomyelin, cholesterol, diacylglycerol, and phosphatidylinositol. Analysis of the obtained electron density profiles and the pressure–distance curves reveals systematic changes in structure and hydration repulsion. The osmotic pressure needed to induce stalk formation at the transition from the fluid lamellar to the rhombohedral phase indicates how membrane fusion properties are modified by bilayer composition
Membrane fusion intermediates and the effect of cholesterol: An in-house X-ray scattering study
No full textWe have developed an X-ray scattering setup which allows to study membrane fusion intermediates or other nonlamellar lipid mesophases by laboratory-scale X-ray sources alone, thus taking advantage of unrestricted beamtime compared to synchrotron sources. We report results of a study of pure lipid bilayers and phospholipid/cholesterol binary mixtures. Stalks, putative intermediate structures occurring during the membrane fusion process, can clearly be identified from reconstructed electron density maps. Phase diagrams of the lyotropic phase behavior of DOPC/cholesterol and DPhPC/cholesterol samples are presented. If cholesterol is present in moderate concentrations, it can substantially promote the formation of stalks at higher degree of hydration. In addition, a possibly new phase in DOPC/cholesterol is found at high cholesterol content in the low humidity range
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