Fig 8 - uploaded by Jonathan D Nickels
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Optimizing experimental conditions for detecting domains in DSPC/DOPC/Chol. The relative homogeneous background scattering Q hom = Q 0 + Q r , calculated from lipid NSLDs (Table 1) using eqn (24)– (29), is plotted vs. fraction of DSPC-d70 (to total DSPC) and the solvent fraction of D 2 O. A global contrast match point is observed at 34.6% D 2 O and 65.9% DSPC-d70 (''CM'', expanded in inset). Close to the contrast match point, Q hom is attenuated by 46 orders of magnitude relative to a fully protiated bilayer in 100% D 2 O solvent (''HC'').
Source publication
In order to understand the biological role of lipids in cell membranes, it is necessary to determine the mesoscopic structure of well-defined model membrane systems. Neutron and X-ray scattering are non-invasive, probe-free techniques that have been used extensively in such systems to probe length scales ranging from angstroms to microns, and dynam...
Contexts in source publication
Context 1
... transition) will exhibit minimal scattering. On the other hand, if the different lipid species segregate from each other into compositionally distinct domains, then neither phase is contrast matched to water, nor are they matched to each other. The resulting spatial contrasts (both lateral and transverse) result in increased scattering. Fig. 8 shows a contour plot of Q hom vs. the fraction of DSPC- d70 (to total DSPC), and the solvent fraction of D 2 O calculated using eqn (24) (Fig. 7, lower left). However, demixing of saturated and unsaturated lipids (i.e., DSPC and DOPC) causes lateral NSLD fluctuations that generate in-plane contrast (Fig. 7, lower right), resulting in ...
Context 2
... (or absence) of domains. At 100% D 2 O (r m = 0.636 fm Å À3 , dashed curves), a large contrast exists between the solvent and bilayer; consequently, the homo- geneous scattering dominates, and there is little apparent difference between uniform (black dashed) and phase- separated (red dashed) vesicles. However, consistent with the prediction of Fig. 8, the differences are greatly magnified near the contrast match point of 34.6% D 2 O (r m = 0.181 fm Å À3 , solid curves). While scattering from a uniformly mixed vesicle exhibits the same relative q-dependence at 100% and 34.6% D 2 O (black dashed and black solid curves, respectively), the total homogeneous intensity is attenuated by a ...
Citations
... Further, antimicrobial peptides or toxins can promote pores in the membranes (Mesa-Galloso et al., 2021). Because of their effects on the SLD profile, all these situations may produce different but characteristic SAS curves (Heberle et al., 2013;Marquardt et al., 2015;Doktorova et al., 2019;Semeraro et al., 2021). ...
Model lipid bilayers have been widely employed as a minimal system to investigate the structural properties of biological membranes by small-angle X-ray (SAXS) and neutron scattering (SANS) techniques. These have nanometre resolution and can give information regarding membrane thickness and scattering length densities (SLDs) of polar and apolar regions. However, biological membranes are complex systems containing different lipids and protein species, in which lipid domains can be dynamically assembled and disassembled. Therefore, SLD variations can occur within the biomembrane. In this work, a novel method has been developed to simulate SAXS and SANS profiles obtained from large unilamellar vesicles containing SLD inhomogeneities that are spatially correlated over the membrane surface. Such inhomogeneities are represented by cylindrical entities with equivalent SLDs. Stacking of bilayers is also included in the model, with no correlation between horizontal and vertical order. The model is applied to a lipid bilayer containing SLD inhomogeneities representing pores, lipid domains, and transmembrane, partially immersed and anchored proteins. It is demonstrated that all the structural information from the host lipid bilayer and from the SLD inhomogeneity can be consistently retrieved by a combined analysis of experimental SAXS and SANS data through the methodology proposed here.
... Given the power of neutron scattering tools for the study of lipid bilayers and the rapidly growing number of publications, there are several prior review papers that also discuss neutron scattering and lipid bilayers as well as introduce the properties of neutrons well. [3][4][5][6][7][8] Among dynamic neutron scattering techniques, triple-axis and time-of-flight (TOF) spectrometers cover energy ranges typically in meV scales (sub ps in time scales), while backscattering (BS) accesses down to 1 leV scales (ns time scales) and neutron spin echo (NSE) techniques accesses tens of neV scales (ls time scales). In lipid membrane dynamics studies, mostly BS and NSE techniques are used to access individual and collective lipid motions, such as rotation, translation, acyl tail motions, and collective membrane fluctuations. ...
Neutron scattering methods are powerful tools for the study of the structure and dynamics of lipid bilayers in length scales from sub Å to tens to hundreds nm and the time scales from sub ps to μs. These techniques also are nondestructive and, perhaps most importantly, require no additives to label samples. Because the neutron scattering intensities are very different for hydrogen- and deuterium-containing molecules, one can replace the hydrogen atoms in a molecule with deuterium to prepare on demand neutron scattering contrast without significantly altering the physical properties of the samples. Moreover, recent advances in neutron scattering techniques, membrane dynamics theories, analysis tools, and sample preparation technologies allow researchers to study various aspects of lipid bilayer dynamics. In this review, we focus on the dynamics of individual lipids and collective membrane dynamics as well as the dynamics of hydration water.
... For instance, lipid head groups shield cholesterol molecules from water, and thus, the ones with large head groups, like sphingomyelins, display higher solubility of cholesterol. 12,13 Saturated lipids are reported to enhance cholesterol solubility as opposed to mono/un-saturated lipids. [14][15][16][17][18] Cholesterol is well known to affect the phase behavior of lipid bilayers. ...
... To a first approximation, pure phospholipid bilayers predominantly display two main phases: ordered and disordered, as distinguished by their spatial order and chain configurational order in the bilayers. 10,12 In the ordered phase, both the spatial and chain configurational order are high. While in the disordered domain, both these orders are low. ...
We show, via molecular simulations, that not only does cholesterol induce a lipid order, but the lipid order also enhances cholesterol localization within the lipid leaflets. Therefore, there is a strong interdependence between these two phenomena. In the ordered phase, cholesterol molecules are predominantly present in the bilayer leaflets and orient themselves parallel to the bilayer normal. In the disordered phase, cholesterol molecules are mainly present near the center of the bilayer at the midplane region and are oriented orthogonal to the bilayer normal. At the melting temperature of the lipid bilayers, cholesterol concentration in the leaflets and the bilayer midplane is equal. This result suggests that the localization of cholesterol in the lipid bilayers is mainly dictated by the degree of ordering of the lipid bilayer. We validate our findings on 18 different lipid bilayer systems, obtained from three different phospholipid bilayers with varying concentrations of cholesterol. To cover a large temperature range in simulations, we employ the Dry Martini force field. We demonstrate that the Dry and the Wet Martini (with polarizable water) force fields produce comparable results.
... Over the last few decades, spatial and dynamic heterogeneity in membrane has been systematically and extensively investigated, in both reconstituted model membranes at carefully chosen composition and cell-derived/living cell membranes (8,10,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). However, our understanding of their functional implications still remains far from complete, mostly due to the lack of a comprehensive molecular level picture. ...
Plasma membrane (PM) heterogeneity has long been implicated in various cellular functions. However, mechanistic principles governing functional regulations of lipid environment are not well understood due to the inherent complexities associated with the relevant length and time scales that limit both direct experimental measurements and their interpretation. In this context, computer simulations hold immense potential to investigate molecular-level interactions and mechanisms that lead to PM heterogeneity and its functions. Herein, we investigate spatial and dynamic heterogeneity in model membranes with coexisting liquid ordered and liquid disordered phases and characterize the membrane order in terms of the local topological changes in lipid environment using the non-affine deformation framework. Furthermore, we probe the packing defects in these membranes , which can be considered as the conjugate of membrane order assessed in terms of the non-affine parameter. In doing so, we formalize the connection between membrane packing and local membrane order and use that to explore the mechanistic principles behind their functions. Our observations suggest that heterogeneity in mixed phase membranes is a consequence of local lipid topology and its temporal evolution, which give rise to disparate lipid packing in ordered and disordered domains. This in turn governs the distinct nature of packing defects in these domains that can play a crucial role in preferential localization of proteins in mixed phase membranes. Furthermore, we observe that lipid packing also leads to contrasting distribution of free volume in the membrane core region in ordered and disordered membranes, which can lead to distinctive membrane permeability of small molecules. Our results, thus, indicate that heterogeneity in mixed phase membranes closely governs the membrane functions, which may emerge from packing-related basic design principles.
... Among indirect techniques, 2 H-NMR (8) or ESR spectra (9) can in favorable cases be decomposed into signals arising from two distinct environments, whereas abrupt changes in FRET between donor and acceptor lipids can occur when phase separation causes spatial reorganization of the probes (10,11,12). With specialized contrastmatching schemes, SANS is sensitive to phase separation of protiated and deuterated lipids (13,14). All indirect methods ultimately involve interpreting an ensemble-averaged signal of limited information content, and most depend on some type of model fitting. ...
Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. This unique application of cryo-EM places particular demands on image acquisition optimization and analysis due to the facts that: 1) each vesicle is a different size with different curvature, 2) the domains in each vesicle can be heterogenous in size and 3) the random orientation of vesicles amplifies the variability of domain size in projected images. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity.
... Over the last few decades, spatial and dynamic heterogeneity in membrane has been systematically and extensively investigated, in both reconstituted model membranes at carefully chosen composition and cell derived/living cell membranes 8,10, [28][29][30][31][32][33][34][35][36][37][38][39] . However, our understanding of their functional implications still remains far from complete, mostly due to the lack of a comprehensive molecular level picture. ...
Plasma membrane (PM) heterogeneity has long been implicated in various cellular functions. However, mechanistic principles governing functional regulations of lipid environment is not well understood due to the inherent complexities associated with the relevant length and time scales that limit both direct experimental measurements and their interpretation. In this context, computer simulation holds immense potential to investigate molecular-level interactions that lead to PM heterogeneity and the related functions. Herein, we investigate spatial and dynamic heterogeneity in model membranes with coexisting liquid ordered and liquid disordered phases and characterize the membrane order in terms of the topological changes in lipid local environment using the non-affine parameter (NAP) frame-work. Furthermore, we probe the packing defects in membrane with coexisting fluid phases, which can be considered as the conjugate of membrane order assessed in terms of the NAP. In doing so, we formalize the connection between membrane packing and local membrane order and use that to explore the mechanistic principles behind preferential localization of proteins in mixed phase membranes and membrane permeability of small molecules. Our observations suggest that heterogeneity in mixed phase membranes follow some generic features, where functions may arise based on packing-related basic design principles.
Significance
Functionally important complex lateral and transverse structures in biological membrane result from the differential molecular interactions among a rich variety of lipids and other building blocks. The nature of molecular packing in membrane is a manifestation of these interactions. In this work, using some of the ideas from the Physics of amorphous materials and glasses, we quantify the correlation between heterogeneous membrane organization and the three dimensional packing defects. Subsequently, we investigate the packing-based molecular design-level features that drive preferential localization of peptides in heterogeneous membrane and membrane permeation of small molecules.
... In the very low-q vector region, the data were not consistent with the fitted model. Two aspects may be considered in explaining the observed intensity oscillation: i) a structure factor contribution accounting for inter-vesicle order, being GM1-containing LUVs charged, and ii) the typical mark coming from the occurrence of GM1-rich patches [56] laterally segregated within the membrane [59], highlighted by the high contrast in the membrane made of deuterated lipids in D 2 O. Combining the SANS and SAXS results, it is plausible that, since Phe does not dampen the low-q oscillations in SANS spectra, the detection of GM1rich patches may be at the origin of the observed features, even if DSC indicates that at the molecular level, the lateral distribution of components is affected in GM1-containing membranes after the interaction with Phe. Nonetheless, a unique data interpretation is then not straightforward and would require investigating the systems at lower q vector values. ...
Phenylketonuria (PKU) is a metabolic disorder connected to an excess of phenylalanine (Phe) in the blood and tissues, with neurological consequences. The disease's molecular bases seem to be related to the accumulation of Phe at the cell membrane surface. Radiological outcomes in the brain demonstrate decreased water diffusivity in white matter, involving axon dysmyelination of not yet understood origin. We used a biophysical approach and model membranes to extend our knowledge of Phe–membrane interaction by clarifying Phe's propensity to affect membrane structure and dynamics based on lipid composition, with emphasis on modulating cholesterol and glycolipid components to mimic raft domains and myelin sheath membranes. Phe showed affinity for the investigated membrane mimics, mainly affecting the Phe-facing membrane leaflet. The surfaces of our neuronal membrane raft mimics were strong anchoring sites for Phe, showing rigidifying effects. From a therapeutic perspective, we further investigated the role of doxycycline, known to disturb Phe packing, unveiling its action as a competitor in Phe interactions with the membrane, suggesting its potential for treatment in the early stages of PKU. Our results suggest how Phe accumulation in extracellular fluids can impede normal growth of myelin sheaths by interfering with membrane slipping and by remodulating free water and myelin-associated water contents.
... Among indirect techniques, 2 H-NMR (8) or ESR spectra (9) can in favorable cases be decomposed into signal arising from two distinct environments, while abrupt changes in FRET between donor and acceptor lipids can occur when phase separation causes spatial reorganization of the probes (10)(11) (12). With specialized contrast-matching schemes, SANS is sensitive to phase separation of protiated and deuterated lipids (13) (14). All indirect methods ultimately involve interpreting an ensemble-averaged signal of limited information content, and most depend on some type of model fitting. ...
Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity.
Significance
Raft domains in unstimulated cells have proven difficult to directly visualize owing to their nanoscopic size and fleeting existence. The few techniques capable of nanoscopic spatial resolution typically rely on interpretation of indirect spectroscopic or scattering signals or require stabilizing the membrane on a solid support. In contrast, cryo-EM yields direct images of nanoscale domains in probe-free, unsupported membranes. Here, we systematically optimize key steps in the experimental and analysis workflow for this new and specialized application. Our findings represent an important step toward developing cryo-EM into a robust method for investigating phase behavior of membranes at length scales relevant to lipid rafts.
... 10,11 In phospholipid environments, glycolipids are known to promote lipid lateral phase separation, 12−15 which is suited, for instance, to locally condensate membrane proteins 12 or to facilitate vesicle budding. 16 The formation of ordered domains in lipid-based model membranes has been investigated with various experimental and computational methods, 17,18 ranging from fluorescence measurements 19 to X-ray/neutron scattering 20−23 and coarse-grained and atomistic molecular dynamics (MD) simulations. 24,25 Grazing-incidence X-ray diffraction (GIXD) 26 is particularly well-suited to study the formation of glycolipid-enriched domains, because the method can identify molecular superlattices which glycolipids in contrast to phospholipids tend to form 27 based on their ability of getting engaged in hydrogen-bond networks (HBNs). ...
Glycolipids are known to be involved in the formation of ordered functional domains in biological membranes. Since the structural characterization of such domains is difficult, most studies have so far dealt with lipid mixtures containing only one glycolipid component at a time, although biological membranes usually contain several glycolipid species, which can result in more complex structures and phase behavior. Here, we combine classical isotherm measurements with surface-sensitive grazing-incidence X-ray diffraction to investigate the phase behavior and miscibility in Langmuir monolayers of binary glycolipid mixtures. We find that the phase behavior has a subtle dependence on the saccharide headgroup chemistry. For compatible chemistries, molecular superlattice structures formed by one of the glycolipid species are conserved and can host foreign glycolipids up to a defined stoichiometry. In contrast, for sterically incompatible saccharide chemistries, the superlattice is lost even if both species are able to form such structures in their pure forms. Our results suggest that related phenomena may play important roles also in biological contexts.
... Cholesterol interaction with phospholipids creates the Lo phase [23]: it increases the order of the fluid phase and decreases the order of the gel phase, resulting in the Lo phase. Cholesterol decouples translational and configurational order, which are both low in the Ld phase and high in the gel phase, such that the Lo phase is characterized by a high configurational order and a low translational one [24]. Cholesterol also plays an essential regulatory function in many biomembrane processes [25], protein and enzyme activities [26,27], and the formation of raft-like domains [28][29][30], in turn associated with cell signalling and intracellular trafficking [31,32]. ...
... The mobility for 14-PCSL is much higher than that measured for 5-PCSL, as expected for the different nitroxide group location, but it is lower than what we measured when handling similar membrane systems but devoid of cholesterol and therefore adopting an Ld phase [22]. Incidentally, the relative low mobility of 14-PCSL can be related to the high conformational order of the acyl phospholipid chains that, in the Lo phase, is similar to the gel phase, while the two phases differ for other features such as lateral mobility of the phospholipids, long-range order, and bilayer hydration [24,60,61]. ...
Lipid structural diversity strongly affects biomembrane chemico-physical and structural properties in addition to membrane-associated events. At high concentrations, cholesterol increases membrane order and rigidity, while polyunsaturated lipids are reported to increase disorder and flexibility. How these different tendencies balance in composite bilayers is still controversial. In this study, electron paramagnetic resonance spectroscopy, small angle neutron scattering, and neutron reflectivity were used to investigate the structural properties of cholesterol-containing lipid bilayers in the fluid state with increasing amounts of polyunsaturated omega-3 lipids. Either the hybrid 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine or the symmetric 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine were added to the mixture of the naturally abundant 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and cholesterol. Our results indicate that the hybrid and the symmetric omega-3 phospholipids affect the microscopic organization of lipid bilayers differently. Cholesterol does not segregate from polyunsaturated phospholipids and, through interactions with them, is able to suppress the formation of non-lamellar structures induced by the symmetric polyunsaturated lipid. However, this order/disorder balance leads to a bilayer whose structural organization cannot be ascribed to either a liquid ordered or to a canonical liquid disordered phase, in that it displays a very loose packing of the intermediate segments of lipid chains.
