Browsing by Author "Porcar, L"
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- ItemApolipoprotein E binding drives structural and compositional rearrangement of mRNA-containing lipid nanoparticles(American Chemical Society (ACS), 2021-03-23) Sebastiani, F; Yanez Arteta, M; Lerche, M; Porcar, L; Lang, C; Bragg, RA; Elmore, CS; Krishnamurthy, VR; Russell, RA; Darwish, TA; Pichler, H; Waldie, S; Moulin, M; Haertlein, M; Forsyth, VT; Lindfors, L; Cárdenas, MEmerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape. © 2021 American Chemical Society. Open Access. This publication is licensed under CC-BY 4.0.
- ItemHuman myelin proteolipid protein structure and lipid bilayer stacking(Springer Nature, 2022-07-12) Ruskamo, S; Raasakka, A; Pedersen, JS; Martel, A; Škubník, K; Darwish, TA; Porcar, L; Kursula, PThe myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure–function differences, as well as fundamental protein–lipid interplay in CNS compact myelin. © 2022 The Authors - Open Access under a Creative Commons Attribution 4.0.
- Item“Invisible” detergents enable a reliable determination of solution structures of native photosystems by small-angle neutron scattering(ACS Publications, 2022-04-06) Golub, M; Gätcke, J; Subramanian, S; Kölsch, A; Darwish, TA; Feoktystov, A; Matsarskaia, O; Martel, A; Porcar, L; Zouni, A; Pieper, JPhotosystems I (PSI) and II (PSII) are pigment–protein complexes capable of performing the light-induced charge separation necessary to convert solar energy into a biochemically storable form, an essential step in photosynthesis. Small-angle neutron scattering (SANS) is unique in providing structural information on PSI and PSII in solution under nearly physiological conditions without the need for crystallization or temperature decrease. We show that the reliability of the solution structure critically depends on proper contrast matching of the detergent belt surrounding the protein. Especially, specifically deuterated (“invisible”) detergents are shown to be properly matched out in SANS experiments by a direct, quantitative comparison with conventional matching strategies. In contrast, protonated detergents necessarily exhibit incomplete matching so that related SANS results systematically overestimate the size of the membrane protein under study. While the solution structures obtained are close to corresponding high-resolution structures, we show that temperature and solution state lead to individual structural differences compared with high-resolution structures. We attribute these differences to the presence of a manifold of conformational substates accessible by protein dynamics under physiological conditions. © 2022 American Chemical Society
- ItemLearning about SANS instruments and data reduction from round robin measurements on samples of polystyrene latex(Wiley Blackwell, 2013-10-01) Rennie, AR; Hellsing, MS; Wood, K; Gilbert, EP; Porcar, L; Schweins, R; Dewhurst, CD; Lindner, P; Heenan, RK; Rogers, SE; Butler, PD; Krzywon, JR; Ghosh, RE; Jackson, AJ; Malfois, MMeasurements of a well-characterized 'standard' sample can verify the performance of an instrument. Typically, small-angle neutron scattering instruments are used to investigate a wide range of samples and may often be used in a number of configurations. Appropriate 'standard' samples are useful to test different aspects of the performance of hardware as well as that of the data reduction and analysis software. Measurements on a number of instruments with different intrinsic characteristics and designs in a round robin can not only better characterize the performance for a wider range of conditions but also, perhaps more importantly, reveal the limits of the current state of the art of small-angle scattering. The exercise, followed by detailed analysis, tests the limits of current understanding as well as uncovering often forgotten assumptions, simplifications and approximations that underpin the current practice of the technique. This paper describes measurements of polystyrene latex, radius 720 angstrom, with a number of instruments. Scattering from monodisperse, uniform spherical particles is simple to calculate and displays sharp minima. Such data test the calibrations of intensity, wavelength and resolution as well as the detector response. Smoothing due to resolution, multiple scattering and polydispersity has been determined. Sources of uncertainty are often related to systematic deviations and calibrations rather than random counting errors. The study has prompted development of software to treat modest multiple scattering and to better model the instrument resolution. These measurements also allow checks of data reduction algorithms and have identified how they can be improved. The reproducibility and the reliability of instruments and the accuracy of parameters derived from the data are described. © 2013, Wiley-Blackwell.
- ItemMeasurement of glucose exclusion from the fully hydrated DOPE inverse hexagonal phase(Royal Society of Chemistry, 2010-03-21) Kent, B; Garvey, CJ; Lenné, T; Porcar, L; Garamus, VM; Bryant, GThe degree of exclusion of glucose from the inverse hexagonal HII phase of fully hydrated DOPE is determined using contrast variation small angle neutron scattering and small angle X-ray scattering. The presence of glucose is found to favour the formation of the non-lamellar HII phase over the fluid lamellar phase, over a wide range of temperatures, while having no significant effect on the structure of the HII phase. Glucose is preferentially excluded from the lipid–water interface resulting in a glucose concentration in the HII phase of less than half that in the coexisting aqueous phase. The degree of exclusion is quantified and the results are consistent with a hydration layer of pure water adjacent to the lipid head groups from which glucose is excluded. The osmotic gradient created by the difference in glucose concentration is determined and the influence of glucose on the phase behaviour of non-lamellar phase forming lipid systems is discussed. © 2010, Royal Society of Chemistry
- ItemNeutron scattering study of a membrane phase miscibility gap: coexistence of L3 "sponge" and La Lamellar phases(Insitute of Physics, 2010-12-16) Hamilton, WA; Porcar, LWe report on a small angle neutron scattering (SANS) study of a temperature driven first order phase transition in a 25wt% solution of the surfactant AOT (Sodium Di-2-ethylhexyl Sulfosuccinate) in 1.5wt% heavy brine between an isotropic L3 "sponge" state at 27°C and a stacked lamellar Lα monophase 55°C. The prominent scattering features of these phases are correlation peaks due to the mean passage size of the L3 sponge and the Lα stacking separation. This ratio of the monophase peak positions Qα/Q31.3, is consistent with previous observations in this and similar systems. In the present study we tracked this system through the intermediate L3 +Lα biphasic miscibility gap. There the initial appearance of the Lα peak at 33.25°C was at a scattering vector some 23% higher than the final high temperature monophase value. During coexistence both L3 and Lα phase peak positions decreased linearly with increasing temperature maintaining a roughly constant ratio Qα/Q3 ~1.6-1.7. Two phase fits to the scattering data and application of scaling law predictions allow us to obtain local L3 phase volume fractions in the biphasic region and make preliminary determinations of the structural accomodations necessitated by phase coexistence in this system's miscibility gap.© 2010, Insitute of Physics
- ItemNew approach to quantification of metamorphism using ultra-small and small angle neutron scattering(Elsevier, 2009-12-15) Anovitz, LM; Lynn, GW; Cole, DR; Rother, G; Allard, LF; Hamilton, WA; Porcar, L; Kim, MHIn this paper we report the results of a study using small angle and ultra-small angle neutron scattering techniques (SANS and USANS) to examine the evolution of carbonates during contact metamorphism. Data were obtained from samples collected along two transects in the metamorphosed Hueco limestone at the Marble Canyon, Texas, contact aureole. These samples were collected from the igneous contact out to similar to 1700 m. Scattering curves obtained from these samples show mass fractal behavior at low scattering vectors, and surface fractal behavior at high scattering vectors. Significant changes are observed in the surface and mass fractal dimensions as well as the correlation lengths (pore and grain sizes), surface area to volume ratio and surface Gibbs Free energy as a function of distance, including regions of the aureole outside the range of classic metamorphic petrology. A change from mass-fractal to non-fractal behavior is observed at larger scales near the outer boundary of the aureole that implies significant reorganization of pore distributions early in the metamorphic history. Surface fractal results suggest significant smoothing of grain boundaries, coupled with changes in pore sizes. A section of the scattering Curve with a slope less than -4 appears Lit low-Q in metamorphosed samples, which is not present in unmetamorphosed samples. A strong spike in the surface area to volume ratio is observed in rocks near the mapped metamorphic limit, which is associated with reaction of small amounts of organic material to graphite. It may also represent an increase in pore Volume or permeability, suggesting that a high permeability zone forms at the boundary of the aureole and moves outwards as metamorphism progresses. Neutron scattering data also correlate well with transmission electron microscopic (TEM) observations, which show formation of micro- and nanopores and microfractures during metamorphism. The scattering data are, however, quantifiable for a bulk rock in a manner that is difficult to achieve using high-resolution imaging (e.g. TEM). Thus, neutron scattering techniques provide a new approach to the analysis and study of metamorphism. © 2009, Elsevier Ltd.
- ItemNew approach to quantification of metamorphism using ultra-small and small angle neutron scattering(Elsevier; Cambridge Publications, 2008-07) Anovitz, LM; Lynn, GW; Cole, DR; Rother, G; Allard, LF; Hamilton, WA; Porcar, L; Kim, MHSmall- and Ultra-Small Angle Neutron Scattering (SANS and USANS) provide powerful tools for analysis of porous rocks because neutrons probe both the surface and the interior of the material providing bulk statistical information over a wide range of length scales. For monomineralic materials scattering contrast arises from the difference between the scattering length density of the rock and the pores (taken to be zero). Pore-grain interfaces are best described by self-similar fractals with non-universal dimensions (2 < D < 3). This leads to a non-integer power-law as a function of the scattering I(Q) = I(1)Q-a + B where B is the incoherent background. For a volume or mass fractal scatterer a = D; if only the surface is a fractal, then a = 6-D. In this manner, surface fractals (a>3) and mass fractals (a<3) are easily distinguished. Non-fractal “fuzzy” interfaces (a>4) may also be observed. We have used SANS and USANS to characterize samples from two transects (contact out to ~1700 m) from the contact metamorphosed Hueco limestone at Marble Canyon, TX. Significant changes in a number of scattering parameters are observed as a function of distance, including regions of the aureole outside the range of classic reaction petrology. Our modeling suggests that changes in surface free energy, pore volume and hydrogen content, among other variables, can be quantified. Both the mass and surface fractals Dm and Ds can be fitted as a function of the surface fractal correlation length as: log10(D/r) = -0.9648 * log10r(Å) + 0.30103 r2 = 0.9999, s = 0.038 although metamorphism does not necessarily proceed linearly along this curve with increasing grade. Nonetheless, the ability to measure these variables shows that these techniques provide a novel approach to the analysis and study of metamorphism.