| 1. |
- Darre, L., et al.
(författare)
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Molecular Dynamics Simulations and Neutron Reflectivity as an Effective Approach To Characterize Biological Membranes and Related Macromolecular Assemblies
- 2015
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Ingår i: Journal of Chemical Theory and Computation. - : American Chemical Society (ACS). - 1549-9618 .- 1549-9626. ; 11:10, s. 4875-4884
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Tidskriftsartikel (refereegranskat)abstract
- In combination with other spectroscopy, microscopy, and scattering techniques, neutron reflectivity is a powerful tool to characterize biological systems. Specular reflection of neutrons provides structural information at the nanometer and subnanometer length scales, probing the composition and organization of layered materials. Currently, analysis of neutron reflectivity data involves several simplifying assumptions about the structure of the sample under study, affecting the extraction and interpretation of information from the experimental data. Computer simulations can be used as a source of structural and dynamic data with atomic resolution. We present a novel tool to compare the structural properties determined by neutron reflectivity experiments with those obtained from molecular simulations. This tool allows benchmarking the ability of molecular dynamics simulations to reproduce experimental data, but it also promotes unbiased interpretation of experimentally determined quantities. Two application examples are presented to illustrate the capabilities of the new tool. The first example is the generation of reflectivity profiles for a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer from molecular dynamics simulations using data from both atomistic and coarse-grained models, and comparison with experimentally measured data. The second example is the calculation of lipid volume changes with temperature and composition from all atoms simulations of single and mixed 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) bilayers.
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| 2. |
- de Ghellinck, Alexis, et al.
(författare)
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Lipid polyunsaturation determines the extent of membrane structural changes induced by Amphotericin B in Pichia pastoris yeast
- 2015
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736. ; 1848:10, s. 2317-2325
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Tidskriftsartikel (refereegranskat)abstract
- The activity of the potent but highly toxic antifungal drug Amphotericin B (AmB), used intravenously to treat systemic fungal and parasitic infections, is widely accepted to result from its specific interaction with the fungal sterol ergosterol. While the effect of sterols on AmB activity has been intensely investigated, the role of membrane phospholipid composition has largely been ignored, and structural studies of native membranes have been hampered by their complex and disordered nature. We show for the first time that the structure of fungal membranes derived from Pichia pastoris yeast depends on the degree of lipid polyunsaturation, which has an impact on the structural consequences of AmB activity. AmB inserts in yeast membranes even in the absence of ergosterol, and forms an extra-membraneous layer whose thickness is resolved to be 4-5 nm. In ergosterol-containing membranes, AmB insertion is accompanied by ergosterol extraction into this layer. The AmB-sponge mediated depletion of ergosterol from P. pastoris membranes gives rise to a significant membrane thinning effect that depends on the degree of lipid polyunsaturation. The resulting hydrophobic mismatch is likely to interfere with a much broader range of membrane protein functions than those directly involving ergosterol, and suggests that polyunsaturated lipids could boost the efficiency of AmB. Furthermore, a low degree of lipid polyunsaturation leads to least AmB insertion and may protect host cells against the toxic effects of AmB. These results provide a new framework based on lipid composition and membrane structure through which we can understand its antifungal action and develop better treatments. (C) 2015 Elsevier B.V. All rights reserved.
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| 3. |
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| 4. |
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| 5. |
- Fisher, Zoe, et al.
(författare)
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Biological Structures
- 2017
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Ingår i: Experimental Methods in the Physical Sciences. - 1079-4042. ; 49, s. 1-75
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Bokkapitel (refereegranskat)abstract
- Neutron scattering methods are excellent for probing the detailed structure of biological systems, which rely on the intricate interplay of a large number of molecules from proteins and nucleic acids to lipids, hormones, and metabolites. With recent instrument developments and emergence of both new neutron sources and techniques, many biological systems that are not yet amenable to characterization by neutron scattering will become accessible in the near future, which will allow new experiments to be developed with a range of biologically relevant samples, offering new insights in life science. In this chapter, we will describe neutron methods for biological structure characterization on different length scales from atomic resolution to macromolecular length scales-up to micrometers. The dynamics of biological molecules are described by Seydel in Chapter 2 of this thematic volume.
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| 6. |
- Lind, Tania Kjellerup, et al.
(författare)
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Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 10:12, s. 10687-10694
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Tidskriftsartikel (refereegranskat)abstract
- Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.
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| 7. |
- Luchini, Alessandra, et al.
(författare)
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The impact of deuteration on natural and synthetic lipids : A neutron diffraction study
- 2018
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Ingår i: Colloids and Surfaces B: Biointerfaces. - : Elsevier BV. - 0927-7765. ; 168, s. 126-133
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Tidskriftsartikel (refereegranskat)abstract
- The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures.
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| 8. |
- Narayanan, Theyencheri, et al.
(författare)
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Recent applications of synchrotron radiation and neutrons in the study of soft matter
- 2017
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Ingår i: Crystallography Reviews. - 0889-311X. ; 23:3, s. 160-226
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Forskningsöversikt (refereegranskat)abstract
- The broad range of applications of synchrotron and neutron scattering in the investigation of soft condensed matter is reviewed. Appropriate combinations of these techniques allow probing the structure and dynamics of these complex systems from sub-nm to micron size scales and picoseconds to seconds and longer time ranges. Applications include a myriad of systems such as polymers, colloids, surfactants, phospholipids, biological macromolecules and functional materials both in bulk and at interfaces. Most studies are performed in situ under the real thermodynamic state of the given system and large ensemble averaged information is readily obtained. The new generations of synchrotron and neutron sources open possibilities for investigating more complex soft matter systems in hitherto unexplored dynamical states.
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| 9. |
- Raasakka, Arne, et al.
(författare)
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Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail
- 2019
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14:6
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Tidskriftsartikel (refereegranskat)abstract
- Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane.
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| 10. |
- Wacklin, Hanna, et al.
(författare)
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Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation.
- 2015
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 0005-2736.
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Tidskriftsartikel (refereegranskat)abstract
- Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII-membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII-lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC-SM-Chol membranes in a manner identical to the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein-membrane interactions available from neutron reflection measurements.
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