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The Effects of Nanoparticle Morphology and Acyl Chain Length on Spontaneous Lipid Transfer Rates
Abstract
We report on studies of lipid transfer rates between different morphology nanoparticles and lipids with different lengths of acyl chains. The lipid transfer rate of dimyristoyl phosphatidylcholine (di-C14, DMPC) in discoidal "bicelles" (0.156 h(-1)) is two orders of magnitude greater than that of DMPC vesicles (ULVs) (1.1 x 10(-3) h(-1)). For both bicellar and ULV morphologies, increasing acyl chain length by two carbons [going from di-C14 DMPC to di-C16, dipalmitoyl phosphatidylcholine (DPPC)], causes lipid transfer rates to decrease by more than two orders of magnitude. Results from small angle neutron scattering (SANS), differential scanning calorimetry (DSC), and fluorescence correlation spectroscopy (FCS) are in good agreement with each other. The present studies highlight the importance of lipid dynamic processes taking place in different morphology biomimetic membranes.
... Lipid transfer proteins are also equipped with membrane binding domains or motifs that may enable them to target donor and acceptor membranes with particular compositions and biophysical properties (10,11). As demonstrated by in vitro experiments, lipids with subtle chemical differences are also passively exchanged between membranes at different rates (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). For example, diacyl phosphatidylcholine (PC) exchanges much more slowly than lysoPC, its single-tailed counterpart (14,20). ...
... For example, diacyl phosphatidylcholine (PC) exchanges much more slowly than lysoPC, its single-tailed counterpart (14,20). Even the addition of just two carbons to an acyl chain of a phospholipid reduces its exchange rate by roughly 10-fold (14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The physical properties and chemical composition of the donor and acceptor membranes additionally influence the rate at which a lipid is passively transported (19)(20)(21)(22)(23)(24)(25)(26). ...
... Even the addition of just two carbons to an acyl chain of a phospholipid reduces its exchange rate by roughly 10-fold (14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The physical properties and chemical composition of the donor and acceptor membranes additionally influence the rate at which a lipid is passively transported (19)(20)(21)(22)(23)(24)(25)(26). For example, dimristoylphosphatidylcholine (DMPC) exchanges more rapidly between liquid-crystalline (L ) phase vesicles than more ordered gel (L ) phase ones (20,26). ...
The collective behavior of lipids with diverse chemical and physical features determines a membrane’s thermodynamic properties. Yet, the influence of lipid physicochemical properties on lipid dynamics, in particular interbilayer transport, remains underexplored. Here, we systematically investigate how the activation free energy of passive lipid transport depends on lipid chemistry and membrane phase. Through all-atom molecular dynamics simulations of 11 chemically distinct glycerophos-pholipids, we determine how lipid acyl chain length, unsaturation, and headgroup influence the free energy barriers for two elementary steps of lipid transport, lipid desorption, which is rate-limiting, and lipid insertion into a membrane. Consistent with previous experimental measurements, we find that lipids with longer, saturated acyl chains have increased activation free energies compared to lipids with shorter, unsaturated chains. Lipids with different headgroups exhibit a range of activation free energies; however, no clear trend based solely on chemical structure can be identified, mirroring difficulties in the interpretation of previous experimental results. Compared to liquid-crystalline phase membranes, gel phase membranes exhibit substantially increased free energy barriers. Overall, we find that the activation free energy depends on a lipid’s local hydrophobic environment in a membrane and that the free energy barrier for lipid insertion depends on a membrane’s interfacial hydrophobicity. Both of these properties can be altered through changes in lipid acyl chain length, lipid headgroup, and membrane phase. Thus, the rate of lipid transport can be tuned through subtle changes in local membrane composition and order, suggesting an unappreciated role for nanoscale membrane domains in regulating cellular lipid dynamics.
SIGNIFICANCE
Cell homeostasis requires spatiotemporal regulation of heterogeneous membrane compositions, in part, through non-vesicular transport of individual lipids between membranes. By systematically investigating how the chemical diversity present in glycerophospholipidomes and variations in membrane order influence the free energy barriers for passive lipid transport, we discover a correlation between the activation free energy and membrane hydrophobicity. By demonstrating how membrane hydrophobicity is modulated by local changes in membrane composition and order, we solidify the link between membrane physicochemical properties and lipid transport rates. Our results suggest that variations in cell membrane hydrophobicity may be exploited to direct non-vesicular lipid traffic.
... [8][9][10] Previous computational work [11][12][13][14][15] on lipid transport has presumed that a lipid's displacement normal to the membrane is the reaction coordinate ( Figure 1A) and has yielded results in conflict with experimental findings. [16][17][18][19] Here we show that the reaction coordinate for passive lipid exchange is indeed more subtle than a simple distance measurement. The reaction coordinate characterizes the creation (or disruption) of a locally hydrophobic environment around the incoming (or outgoing) lipid ( Figure 1B). ...
... Based on experimentally measured activation energies, calculated activation free energies for lipid exchange exceed free energies for transferring a lipid from water to a membrane, indicating that there is a barrier for lipid desorption and insertion. [16][17][18][19] At this barrier, the desorbing lipid is hypothesized to only have the terminal carbons of its tails left within the membrane. 16,27 Thus, the activation free energy required to form the transition state has been attributed to the creation of a cavity in the membrane due to partial removal of a lipid and another cavity in the solvent to accommodate that lipid. ...
... [11][12][13][14][15] These free energy profiles lack a barrier for insertion, 11-15 seemingly in conflict with experimental results. [16][17][18][19] If the COM displacement is not the reaction coordinate for lipid exchange, the kinetically relevant barrier may not be resolved in these free energy profiles; instead, a barrier may exist along a different degree of freedom that is the reaction coordinate. Consistent with this idea, Vermaas and Tajkhorshid's MD study of lipid insertion indicated that the COM displacement is not sufficient to fully describe the microscopic dynamics of lipid insertion. ...
The maintenance of heterogeneous lipid compositions among cellular membranes is key to biological function. Yet, even the simplest process that could be responsible for maintaining proper lipid distributions, passive lipid exchange of individual molecules between membranes, has eluded a detailed understanding, due in part to inconsistencies between experimental findings and molecular simulations. We resolve these discrepancies by discovering the reaction coordinate for passive lipid exchange, which enables a complete biophysical characterization of the rate limiting step for lipid exchange. Our approach to identify the reaction coordinate capitalizes on our ability to harvest over 1,000 unbiased trajectories of lipid insertion, an elementary step of passive lipid transport, using all-atom and coarse-grained molecular dynamics simulations. We find that the reaction coordinate measures the formation and breakage of hydrophobic contacts between the membrane and exchanging lipid. Consistent with experiments, free energy profiles as a function of our reaction coordinate exhibit a substantial barrier for insertion. In contrast, lipid insertion was predicted to be a barrier-less process by previous computational studies, which incorrectly presumed the reaction coordinate to be the displacement of the exchanging lipid from the membrane. Utilizing our newfound knowledge of the reaction coordinate, we formulate an expression for the lipid exchange rate to enable a quantitative comparison with experiments. Overall, our results indicate that the breakage of hydrophobic contacts is rate limiting for passive lipid exchange and provide a foundation to understand the catalytic function of lipid transfer proteins.
... However, the apparent rate k app did not depend significantly on the PC concentration in the liposomes for HDL (Fig. S6C). The fraction of lipids that do not undergo exchange in the observed time range, f tight , probably reflects the presence of lipids with longer tail groups (see lipidomics in Fig. 6, where the main lipid species are given) as the unimer expulsion rate is critically dependent on the hydrophobic length of the amphiphiles [39][40][41] . For example, Nieh and coworkers 41 have found a decrease in lipid transfer rates by more than two orders of magnitude when the acyl chain length was increased by only two carbons (from di-C14 DMPC to di-C16 (DPPC)) in bicelles and vesicles. ...
... The fraction of lipids that do not undergo exchange in the observed time range, f tight , probably reflects the presence of lipids with longer tail groups (see lipidomics in Fig. 6, where the main lipid species are given) as the unimer expulsion rate is critically dependent on the hydrophobic length of the amphiphiles [39][40][41] . For example, Nieh and coworkers 41 have found a decrease in lipid transfer rates by more than two orders of magnitude when the acyl chain length was increased by only two carbons (from di-C14 DMPC to di-C16 (DPPC)) in bicelles and vesicles. Thus, it is likely that lipids with longer tail groups are not readily exchanged. ...
... The first kinetic regime for LDL can be explained by a rate of inter-particle lipid exchange and shows the same order of magnitude as previously observed for other PC liposomes in the fluid phase 41,42 . In lipoproteins, the phospholipids are constrained to a monolayer as opposed to the bicelle, nanodisc and other vesicular structures 42 . ...
Atherosclerosis is the main killer in the western world. Today’s clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, “neutron invisible” phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.
... Based on crystallographic measurements, a cleft-like gating mechanism has been proposed to control access to CPTP's hydrophobic cavity and to facilitate C1P entry and exit [16]; however, because such molecular motions have yet to be resolved, their importance for C1P uptake and release is unclear. An additional puzzle is how CPTP overcomes the significant free energy barrier for lipid desorption from a membrane, which limits passive lipid transport rates [21][22][23][24][25]. We recently demonstrated that the activation free energy barrier for passive lipid desorption reflects the thermodynamic cost of breaking hydrophobic lipid-membrane contacts [25], and that it can be lowered by decreasing the hydrophobicity of the transferring lipid's local membrane environment [26]. ...
... In in vitro lipid transfer assays, CPTP transfers approximately 4 C1P molecules per minute [16,19,20]. While the rate of passive C1P transport is expected to be at least two orders-of-magnitude slower [19][20][21][22][23][24], to our knowledge, it has not yet been measured experimentally, hindering a quantitative comparison between our simulations and experiment. As determined in our previous studies [25,26], both simulation and experiment yield the same values for changes in the activation barrier for passive lipid transport, for example due to differences in membrane physicochemical properties, even though the absolute barrier height is severely underestimated in simulation. ...
Cellular distributions of the sphingolipid ceramide-1-phosphate (C1P) impact essential biological processes. C1P levels are spatiotemporally regulated by ceramide-1-phosphate transfer protein (CPTP), which efficiently shuttles C1P between organelle membranes. Yet, how CPTP rapidly extracts and inserts C1P into a membrane remains unknown. Here, we devise a multiscale simulation approach to elucidate biophysical details of CPTP-mediated C1P transport. We find that CPTP binds a membrane poised to extract and insert C1P and that membrane binding promotes conformational changes in CPTP that facilitate C1P uptake and release. By significantly disrupting a lipid’s local hydrophobic environment in the membrane, CPTP lowers the activation free energy barrier for passive C1P desorption and enhances C1P extraction from the membrane. Upon uptake of C1P, further conformational changes may aid membrane unbinding in a manner reminiscent of the electrostatic switching mechanism used by other lipid transfer proteins. Insertion of C1P into an acceptor membrane, eased by a decrease in membrane order by CPTP, restarts the transfer cycle. Most notably, we provide molecular evidence for CPTP’s ability to catalyze C1P extraction by breaking hydrophobic C1P–membrane contacts with compensatory hydrophobic lipid–protein contacts. Our work, thus, provides biophysical insights into how CPTP efficiently traffics C1P between membranes to maintain sphingolipid homeostasis and, additionally, presents a simulation method aptly suited for uncovering the catalytic mechanisms of other lipid transfer proteins.
... Our multiscale simulation approach offers a framework to efficiently test this hypothesis and, thus, further our molecular knowledge of how lipid transfer proteins function to regulate cellular lipid distributions. explains why CPTP facilitated transport of C1P is more rapid than passive transport, it 24 fails to explain how CPTP swiftly extracts (and inserts) C1P from (into) a membrane. 25 Based on crystallographic measurements, a cleft-like gating mechanism has been 26 proposed to control access to CPTP's hydrophobic cavity and to facilitate C1P entry 27 and exit [16]; however, because such molecular motions have yet to be resolved, their 28 importance for C1P uptake and release is unclear. ...
... 25 Based on crystallographic measurements, a cleft-like gating mechanism has been 26 proposed to control access to CPTP's hydrophobic cavity and to facilitate C1P entry 27 and exit [16]; however, because such molecular motions have yet to be resolved, their 28 importance for C1P uptake and release is unclear. An additional puzzle is how CPTP 29 overcomes the significant free energy barrier for lipid desorption from a membrane, 30 which limits passive lipid transport rates [21][22][23][24][25]. We recently demonstrated that the 31 activation free energy barrier for passive lipid desorption reflects the thermodynamic 32 cost of breaking hydrophobic lipid-membrane contacts [25], and that it can be lowered 33 by decreasing the hydrophobicity of the transferring lipid's local membrane 34 environment [26]. ...
Cellular distributions of the sphingolipid ceramide-1-phosphate (C1P) impact essential biological processes. C1P levels are spatiotemporally regulated by ceramide-1-phosphate transfer protein (CPTP), which efficiently shuttles C1P between organelle membranes. Yet, how CPTP rapidly extracts and inserts C1P into a membrane remains unknown. Here, we devise a multiscale simulation approach to elucidate the biophysical details of CPTP-mediated C1P transport. We find that CPTP binds a membrane poised to extract and insert C1P and that membrane binding promotes conformational changes in CPTP that facilitate C1P uptake and release. By substantially disrupting a lipid’s local hydrophobic environment in the membrane, CPTP lowers the activation free energy barrier for passive C1P desorption and enhances C1P extraction from the membrane. Upon uptake of C1P, further conformational changes may aid membrane unbinding in a manner reminiscent of the electrostatic switching mechanism used by other lipid transfer proteins. Insertion of C1P into an acceptor membrane, eased by a decrease in membrane order by CPTP, restarts the transfer cycle. Most notably, we provide molecular evidence for CPTP’s ability to catalyze C1P extraction by breaking hydrophobic C1P–membrane contacts with compensatory hydrophobic lipid–protein contacts. Our work, thus, provides biophysical insights into how CPTP efficiently traffics C1P between membranes to maintain sphingolipid homeostasis and, additionally, presents a simulation method aptly suited for uncovering the catalytic mechanisms of other lipid transfer proteins.
Author summary
Critical cellular processes require spatiotemporal regulation of sphingolipid levels among organelle membranes. Programmed cell death and inflammation, for example, are impacted by the distribution of ceramide-1-phosphate (C1P). C1P levels are specifically altered by ceramide-1-phosphate transfer protein (CPTP), which mediates C1P intermembrane transport. Using a multiscale simulation approach tailored to studying lipid transport, we elucidate the molecular mechanism used by CPTP to rapidly transport C1P between membranes: Through conformational changes that are coupled to membrane binding, CPTP significantly disrupts C1P’s local hydrophobic environment in a membrane and catalyzes its extraction. Since this catalytic mechanism is biophysically related to that of passive lipid transport, it may be ubiquitously used by lipid transport proteins to rapidly traffic lipids between membranes and ensure membrane homeostasis. Our multiscale simulation approach offers a framework to efficiently test this hypothesis and, thus, further our molecular knowledge of how lipid transfer proteins function to regulate cellular lipid distributions.
... As research on plant growth in soil polluted by nano-TiO 2 has shown that plants can absorb and transport nano-TiO 2 from the root to the leaf [8,9], studies on the toxic effects of nano-TiO 2 in plants is very important. For example, nano-TiO 2 possess certain photocatalytic properties, causing damage to plants by inducing reactive oxygen species (ROS) production [10][11][12][13][14]. Moreover, accumulating evidence has verified the toxic effects of nano-TiO 2 on plants, causing DNA damage in Allium cepa roots and Nicotiana tabacum leaves, also with excessive lipid peroxidation products detected in A. cepa roots [15]. ...
... Nonetheless, it remains unclear how nano-TiO 2 activate expression of PIN2, YUC8 and TIR1. Previous studies have reported that nano-TiO 2 induce ROS production [10][11][12]14], and auxin-related genes are regulated by H 2 O 2 [31]. However, it remains to be further explored how nano-TiO 2 modulate the expression of auxin-related genes involved in root growth. ...
... As research on plant growth in soil polluted by nano-TiO 2 has shown that plants can absorb and transport nano-TiO 2 from the root to the leaf [8,9], studies on the toxic effects of nano-TiO 2 in plants is very important. For example, nano-TiO 2 possess certain photocatalytic properties, causing damage to plants by inducing reactive oxygen species (ROS) production [10][11][12][13][14]. Moreover, accumulating evidence has verified the toxic effects of nano-TiO 2 on plants, causing DNA damage in Allium cepa roots and Nicotiana tabacum leaves, also with excessive lipid peroxidation products detected in A. cepa roots [15]. ...
... Nonetheless, it remains unclear how nano-TiO 2 activate expression of PIN2, YUC8 and TIR1. Previous studies have reported that nano-TiO 2 induce ROS production [10][11][12]14], and auxin-related genes are regulated by H 2 O 2 [31]. However, it remains to be further explored how nano-TiO 2 modulate the expression of auxin-related genes involved in root growth. ...
Comic character detection is becoming an exciting and growing research area in the domain of machine learning. In this regard, recently, many methods are proposed to provide adequate performance. However, most of these methods utilized the custom datasets, containing a few hundred images and fewer classes, to evaluate the performances of their models without comparing it, with some standard datasets. This article takes advantage of utilizing a standard publicly dataset taken from a competition, and proposes a generic data balancing technique for imbalanced dataset to enhance and enable the in-depth training of the CNN. In addition, to classify the superheroes efficiently, a custom 17-layer deep convolutional neural network is also proposed. The computed results achieved overall classification accuracy of 97.9% which is significantly superior to the accuracy of competition’s winner.
... These investigations provided insight into the effect of cholesterol on flip-flop and found a much slower rate than previously reported [81]. In another study, it was shown that the acyl (tail) length of the lipid plays a crucial role in controlling the timescale of exchange [83] analogous to polymeric surfactants [77]. The chain packing in different morphologies, vesicles or discs was also found to influence the kinetics due to changes in the lipid packing [83]. ...
... In another study, it was shown that the acyl (tail) length of the lipid plays a crucial role in controlling the timescale of exchange [83] analogous to polymeric surfactants [77]. The chain packing in different morphologies, vesicles or discs was also found to influence the kinetics due to changes in the lipid packing [83]. Although these studies have substantially contributed to the understanding of lipid membrane dynamics, there is considerable work remaining to explore the dynamics and associated signalling events in cells which are complex mixtures of many lipids and other biomolecules. ...
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.
... With this method, the cholesterol headgroup and bilayer tails can be located by deuterating either species (Harroun et al., 2006). The contrast matching strategy has also been successfully implemented to probe the dynamical properties of membranes such as the flip-flop of lipids between two leaflets in a bilayer and inter-particle lipid and cholesterol transfer (Xia et al., 2015;Xia et al., 2016;Nakano et al., 2007;Nakano et al., 2009). One important application of model membranes is for reconstitution of membrane proteins in order to preserve the membrane function and structure. ...
Spectroscopic, scattering, and imaging methods play an important role in advancing the study of pharmaceutical and biopharmaceutical therapies. The tools more familiar to scientists within industry and beyond, such as nuclear magnetic resonance and fluorescence spectroscopy, serve two functions: as simple high-throughput techniques for identification and purity analysis, and as potential tools for measuring dynamics and structures of complex biological systems, from proteins and nucleic acids to membranes and nanoparticle delivery systems. With the expansion of commercial small-angle x-ray scattering instruments into the laboratory setting and the accessibility of industrial researchers to small-angle neutron scattering facilities, scattering methods are now used more frequently in the industrial research setting, and probe-less time-resolved small-angle scattering experiments are now able to be conducted to truly probe the mechanism of reactions and the location of individual components in complex model or biological systems. The availability of atomic force microscopes in the past several decades enables measurements that are, in some ways, complementary to the spectroscopic techniques, and wholly orthogonal in others, such as those related to nanomechanics. As therapies have advanced from small molecules to protein biologics and now messenger RNA vaccines, the depth of biophysical knowledge must continue to serve in drug discovery and development to ensure quality of the drug, and the characterization toolbox must be opened up to adapt traditional spectroscopic methods and adopt new techniques for unraveling the complexities of the new modalities. The overview of the biophysical methods in this review is meant to showcase the uses of multiple techniques for different modalities and present recent applications for tackling particularly challenging situations in drug development that can be solved with the aid of fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, atomic force microscopy, and small-angle scattering.
... The experimental method and analysis of the data is described in detail in the supplementary information. Previously, it has been shown that the exchange and flip-flop rates are directly dependent on the acyl chain length due to changes in solubility and fluidity [69,70]. All the experiments presented are done at temperatures above the melting temperature of the lipids (above 24 degrees) as the membrane is then in the fluid liquid crystalline phase. ...
Hypothesis: Most textbook models for antimicrobial peptides (AMP) mode of action are focused on structural effects and pore formation in lipid membranes, while these deformations have been shown to require high concentrations of peptide bound to the membrane. Even insertion of low amounts of peptides in the membrane is hypothesized to affect the transmembrane transport of lipids, which may play a key role in the peptide effect on membranes.
Experiments: Here we combine state-of-the-art small angle X-ray/neutron scattering (SAXS/SANS) techniques to systematically study the effect of a broad selection of natural AMPs on lipid membranes. Our approach enables us to relate the structural interactions, effects on lipid exchange processes, and thermodynamic parameters, directly in the same model system.
Findings: The studied peptides, indolicidin, aurein 1.2, magainin II, cecropin A and LL-37 all cause a general acceleration of essential lipid transport processes, without necessarily altering the overall structure of the lipid membranes or creating organized pore-like structures. We observe rapid scrambling of the lipid composition associated with enhanced lipid transport which may trigger lethal signaling processes and enhance ion transport. The reported membrane effects provide a plausible canonical mechanism of AMP-membrane interaction and can reconcile many of the previously observed effects of AMPs on bacterial membranes.
... This implies that the "rim" of discoidal bicelle may not have ideal, clear boundary as depicted in the schematic of Fig 2. A more realistic portrayal of the rim should contain a less-dense mixture of DPPC and DHPC (in contrast to gel-phase planar DPPC), both of which are presumably in the Lα phase and/or loosely associated with water, resulting in the distinct contrast between rim and other part regions of bicelles. This explanation agrees with a recent report indicating that the lipid transfer rate of DPPC between bicelles is two orders of magnitude higher than that between liposomes (Xia et al., 2015(Xia et al., , 2016. The enhanced lipid transfer rate was presumably attributed to the DPPC at vicinity of the rim, consistent with the large loosely-packed "rim" region obtained from this study. ...
The internal profile across the bilayer reveals important structural information regarding the crystallinity of acyl chains or the positions of encapsulated species. Here, we demonstrate that a simple five-layer-core-shell discoidal model can be employed to best fit the extended-q small angle X-ray scattering (SAXS) data and resolve the bilayer internal structure (with sub-nanometer resolution) of a nanoscale discoidal system comprised of a mixture of long- and short- chain lipids (known as “bicelles”). In contrast to the traditional core-shell discoidal model, the detailed structure in the hydrophobic core such as the methylene and methyl groups can be distinguished via this model. The refined model is validated by the SAXS data of bicelles whose electron scattering length density of the hydrophobic core is adjusted by the addition of a long-chain lipid with a fluorine-end group. The higher resolution of the bilayer internal structure can be employed to advance our understanding of the interaction and conformation of the membrane and associated molecules, such as membrane-associated proteins and locations of entrapped species in the lipid nanoparticles.
... Even faster kinetics have been reported for TX-100 micelles (k obs = 1.5 × 10 5 s −1 ). By contrast, DMPC exchange among gel-phase bicelles 35 is slower than lipid transfer in any of the above fluid-phase systems. ...
Some styrene/maleic acid (SMA) copolymers solubilise membrane lipids and proteins to form polymer-bounded nanodiscs termed SMA/lipid particles (SMALPs). Although SMALPs preserve a lipid-bilayer core, they appear to be more dynamic than other membrane mimics. We used time-resolved Förster resonance energy transfer and small-angle neutron scattering to determine the kinetics and the mechanisms of phospholipid transfer among SMALPs. In contrast with vesicles or protein-bounded nanodiscs, SMALPs exchange lipids not only by monomer diffusion but also by fast collisional transfer. Under typical experimental conditions, lipid exchange occurs within seconds in the case of SMALPs but takes minutes to days in the other bilayer particles. The diffusional and second-order collisional exchange rate constants for SMALPs at 30 °C are kdif = 0.287 s−1 and kcol = 222 M−1s−1, respectively. Together with the fast kinetics, the observed invariability of the rate constants with probe hydrophobicity and the moderate activation enthalpy of ~70 kJ mol−1 imply that lipids exchange through a “hydrocarbon continuum” enabled by the flexible nature of the SMA belt surrounding the lipid-bilayer core. Owing to their fast lipid-exchange kinetics, SMALPs represent highly dynamic equilibrium rather than kinetically trapped membrane mimics, which has important implications for studying protein/lipid interactions in polymer-bounded nanodiscs.
... Thus, it has been shown that removing two methylene units from or adding a double bond to an acyl chain of a diacyl GPL increases the rate of diffusion 5-to 10-fold. [18][19][20] Consequently, the rate of intervesicle diffusion of two-chain GPLs can differ by more than 1000-fold (ie, the half-time varies from minutes to days), depending on the number of alkyl chain carbons and double bonds. On the other hand, the polar head group structure has only a modest effect on SLT. ...
In most reviews addressing intracellular lipid trafficking, spontaneous diffusion of lipid monomers between the cellular organelles is considered biologically irrelevant because it is thought to be far too slow to significantly contribute to organelle biogenesis. This view is based on intervesicle transfer experiments carried out in vitro with few lipids as well as on the view that lipids are highly hydrophobic and thus cannot undergo spontaneous intermembrane diffusion at a significant rate. However, besides that single-chain lipids can translocate between vesicles in seconds, it has been demonstrated that the rate of spontaneous transfer of two-chain polar lipids can vary even 1000-fold, depending on the number of carbons and double bonds in the acyl chains. In addition, the rate of spontaneous lipid transfer can strongly depend on the experimental conditions such as vesicle composition and concentration. This review examines the studies suggesting that spontaneous lipid transfer is probably more relevant to intracellular trafficking of amphipathic lipids than commonly thought.
Bicellar systems have become popularized as their rich morphology can be applied in biochemistry, physical chemistry, and drug delivery technology. To the biochemical field, bicelles are powerful model membranes for the study of transmembrane protein behavior, membrane transport, and environmental interactions with the cell. Their morphological responses to environmental changes reveal a profound fundamental understanding of physical chemistry related to the principle of self-assembly. Recently, they have also drawn significant attention as theranostic nanocarriers in biopharmaceutical and diagnostic research due to their superior cellular uptake compared to liposomes. It is evident that applications are becoming broader, demanding to understand how the bicelle will form and behave in various environments. To consolidate current works on the bicelle's modern applications, this review will discuss various effects of composition and environmental conditions on the morphology, phase behavior, and stability. Furthermore, various applications such as payload entrapment and polymerization templating are presented to demonstrate their versatility and chemical nature.
Understanding how lipid dynamics change with membrane curvature is important given that biological membranes constantly change their curvature and morphology through membrane fusion and endo-/exocytosis. Here, we used time-resolved small-angle neutron scattering and time-resolved fluorescence to characterize the properties and dynamics of phospholipids in vesicles with different curvatures. Dissociation of phospholipids from vesicles required traversing an energy barrier comprising positive enthalpy and negative entropy. However, lipids in membranes with high positive curvature have dense acyl chain packing and loose headgroup packing, leading to hydrophobic hydration due to water penetration into the membrane. These properties were found to lower the hydrophobic hydration enhancement associated with phospholipid dissociation and mitigate the acyl chain packing of lipids adjacent to the space created by the lipid dissociation, resulting in an increase in activation entropy. The results of this study provide important insights into the functions of biomembranes in relation to their dynamic structural changes.
Differential scanning calorimetry (DSC) of dipalmitoyl phosphatidylcholine (DPPC), dihexanoyl phosphatidylcholine, and dipalmitoyl phosphatidylglycerol bicelles reveals two endothermic peaks. Based on analysis of small angle neutron scattering and small angle X-ray scattering data, the two DSC peaks are associated with the melting of DPPC and a change in bicellar morphology─namely, either bicelle-to-spherical vesicle or oblate-to-spherical vesicle. The reversibility of the two structural transformations was examined by DSC and found to be consistent with the corresponding small angle scattering data. However, the peak that is not associated with the melting of DPPC does not correspond to any structural transformation for bicelles containing distearoyl phosphatidylethanolamine conjugated with polyethylene glycol. Based on complementary experimental data, we conclude that membrane flexibility, lipid miscibility, and differential solubility between the long- and short-chain lipids in water are important parameters controlling the reversibility of morphologies experienced by the bicelles.
It is well-known that lipids constituting the cytoplasmic membrane undergo continuous reorganization to maintain the appropriate composition important for the integrity of the cell. The transport of lipids is controlled by mainly membrane proteins, but also spontaneous lipid transport between leaflets, lipid “flip–flop”, occurs. These processes do not only occur spontaneously under equilibrium, but also promote structural rearrangements, morphological transitions, and growth processes. It has previously been shown that intravesicular lipid “flip–flop” and intervesicular lipid exchange under equilibrium can be deduced indirectly from contrast variation time-resolved small-angle neutron scattering (TR-SANS) where the molecules are “tagged” using hydrogen/deuterium (H/D) substitution. In this work, we show that this technique can be extended to simultaneously detect changes in the growth and the lipid “flip–flop” and exchange rates induced by a peptide additive on lipid vesicles consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), d-DMPC (1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and small amounts of DMPE-PEG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). Changes in the overall size were independently monitored using dynamic light scattering (DLS). We find that the antimicrobial peptide, indolicidin, accelerates lipid transport and additionally induces limited vesicular growth. Moreover, in TR-SANS experiments using partially labeled lipid mixtures to separately study the kinetics of the lipid components, we show that, whereas peptide addition affects both lipids similarly, DMPG exhibits faster kinetics. We find that vesicular growth is mainly associated with peptide-mediated lipid reorganization that only slightly affects the overall exchange kinetics. This is confirmed by a TR-SANS experiment of vesicles preincubated with peptide showing that after pre-equilibration the kinetics are only slightly slower.
This review focuses on time-resolved neutron scattering, particularly time-resolved small angle neutron scattering (TR-SANS), as a powerful in situ noninvasive technique to investigate intra- and intermembrane transport and distribution of lipids and sterols in lipid membranes. In contrast to using molecular analogues with potentially large chemical tags that can significantly alter transport properties, small angle neutron scattering relies on the relative amounts of the two most abundant isotope forms of hydrogen: protium and deuterium to detect complex membrane architectures and transport processes unambiguously. This review discusses advances in our understanding of the mechanisms that sustain lipid asymmetry in membranes—a key feature of the plasma membrane of cells—as well as the transport of lipids between membranes, which is an essential metabolic process.
The collective behavior of lipids with diverse chemical and physical features determines a membrane’s thermodynamic properties. Yet, the influence of lipid physicochemical properties on lipid dynamics, in particular interbilayer transport, remains underexplored. Here, we systematically investigate how the activation free energy of passive lipid transport depends on lipid chemistry and membrane phase. Through all-atom molecular dynamics simulations of 11 chemically distinct glycerophospholipids, we determine how lipid acyl chain length, unsaturation, and headgroup influence the free energy barriers for two elementary steps of lipid transport: lipid desorption, which is rate limiting, and lipid insertion into a membrane. Consistent with previous experimental measurements, we find that lipids with longer, saturated acyl chains have increased activation free energies compared to lipids with shorter, unsaturated chains. Lipids with different headgroups exhibit a range of activation free energies; however, no clear trend based solely on chemical structure can be identified, mirroring difficulties in the interpretation of previous experimental results. Compared to liquid-crystalline phase membranes, gel phase membranes exhibit substantially increased free energy barriers. Overall, we find that the activation free energy depends on a lipid’s local hydrophobic environment in a membrane and that the free energy barrier for lipid insertion depends on a membrane’s interfacial hydrophobicity. Both of these properties can be altered through changes in lipid acyl chain length, lipid headgroup, and membrane phase. Thus, the rate of lipid transport can be tuned through subtle changes in local membrane composition and order, suggesting an unappreciated role for nanoscale membrane domains in regulating cellular lipid dynamics.
The authors designed a structurally stable nano‐in‐nano (NANO²) system highly capable of bioimaging via an aggregation‐enhanced NIR excited emission and photoacoustic response achieved based on atomically precise gold nanoclusters protected by linear thiolated ligands [Au25(SCnH2n+1)18, n = 4–16] encapsulated in discoidal phospholipid bicelles through a one‐pot synthesis. The detailed morphological characterization of NANO² is conducted using cryogenic transmission electron microscopy, small/wide angle X‐ray scattering with the support of molecular dynamics simulations, providing information on the location of Au nanoclusters in NANO². The photoluminescence observed for NANO² is 20–60 times more intense than that of the free Au nanoclusters, with both excitation and emission wavelengths in the near‐infrared range, and the photoacoustic signal is more than tripled. The authors attribute this newly discovered aggregation‐enhanced photoluminescence and photoacoustic signals to the restriction of intramolecular motion of the clusters’ ligands. With the advantages of biocompatibility and high cellular uptake, NANO² is potentially applicable for both in vitro and in vivo imaging, as the authors demonstrate with NIR excited emission from in vitro A549 human lung and the KB human cervical cancer cells.
Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as ‘good’ and ‘bad’ cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as when using an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.
The maintenance of heterogeneous lipid compositions among cellular membranes is key to biological function. Yet, even the simplest process that could be responsible for maintaining proper lipid distributions, passive lipid exchange of individual molecules between membranes, has eluded a detailed understanding, due in part to inconsistencies between experimental findings and molecular simulations. We resolve these discrepancies by discovering the reaction coordinate for passive lipid exchange, which enables a complete biophysical characterization of the rate-limiting step for lipid exchange. Our approach to identify the reaction coordinate capitalizes on our ability to harvest over 1000 unbiased trajectories of lipid insertion, an elementary step of passive lipid transport, using all-atom and coarse-grained molecular dynamics simulations. We find that the reaction coordinate measures the formation and breakage of hydrophobic contacts between the membrane and exchanging lipid. Consistent with experiments, free energy profiles as a function of our reaction coordinate exhibit a substantial barrier for insertion. In contrast, lipid insertion was predicted to be a barrier-less process by previous computational studies, which incorrectly presumed the reaction coordinate to be the displacement of the exchanging lipid from the membrane. Utilizing our newfound knowledge of the reaction coordinate, we formulate an expression for the lipid exchange rate to enable a quantitative comparison with experiments. Overall, our results indicate that the breakage of hydrophobic contacts is rate limiting for passive lipid exchange and provide a foundation to understand the catalytic function of lipid transfer proteins.
In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently-labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with preparation of vesicles and their rather short ‘shelf-life’. Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different GlycoLipid Transfer Protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol-injection. Among the notable advantages of using bicelle-generated model membranes are: (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation; and (ii) long 'shelf-life' after production (≥ 6 days) and resilience to freeze-thaw storage. The bicelle-dilution based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs, but also for LTP activity detection in crude cytosolic fractions of cell homogenates.
As a novel member of the two-dimensional nanomaterial family, mono- or few-layer black phosphorus (BP) with direct bandgap and high charge carrier mobility is promising in many applications such as microelectronic devices, photoelectronic devices, energy technologies, and catalysis agents. Due to its benign elemental composition (phosphorus), large surface area, electronic/photonic performances, and chemical/biological activities, BP has also demonstrated a great potential in biomedical applications including biosensing, photothermal/photodynamic therapies, controlled drug releases, and antibacterial uses. The nature of the BP–bio interface is comprised of dynamic contacts between nanomaterials (NMs) and biological systems, where BP and the biological system interact. The physicochemical interactions at the nano–bio interface play a critical role in the biological effects of NMs. In this review, we discuss the interface in the context of BP as a nanomaterial and its unique physicochemical properties that may affect its biological effects. Herein, we comprehensively reviewed the recent studies on the interactions between BP and biomolecules, cells, and animals and summarized various cellular responses, inflammatory/immunological effects, as well as other biological outcomes of BP depending on its own physical properties, exposure routes, and biodistribution. In addition, we also discussed the environmental behaviors and potential risks on environmental organisms of BP. Based on accumulating knowledge on the BP–bio interfaces, this review also summarizes various safer-by-design strategies to change the physicochemical properties including chemical stability and nano–bio interactions, which are critical in tuning the biological behaviors of BP. The better understanding of the biological activity of BP at BP–bio interfaces and corresponding methods to overcome the challenges would promote its future exploration in terms of bringing this new nanomaterial to practical applications.
Molecular transfer between nanoparticles has important implication on nanoparticle stability. Recently, the interparticle spontaneous lipid transfer rate constant for discoidal bicelles was found to be very different from spherical, unilamellar vesicles (ULVs). Here we investigate the mechanism responsible for this discrepancy. Analysis of the data indicates that lipid transfer is entropically favorable, but enthalpically unfavorable, with an activation energy that is independent of bicelle size and long- to short-chain lipid molar ratio. Moreover, molecular dynamics simulations reveal a lower lipid dissociation energy cost in the vicinity of interfaces (“defects”) induced by the segregation of long- and short- chain lipids in bicelles – these defects are not present in ULVs. Taken together, these results suggest that the enhanced lipid transfer observed in bicelles arises from interfacial defects as a result of the hydrophobic mismatch between long- and short-chain lipid species. Finally, the observed transfer rate is found to be independent of nanoparticle stability.
Phosphatidylserine (PS) lipids play essential roles in biological processes, including enzyme activation and apoptosis. We report on the molecular structure and atomic scale interactions of a fluid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS). A scattering density profile model, aided by molecular dynamics (MD) simulations, was developed to jointly refine different contrast small-angle neutron and X-ray scattering data, which yielded a lipid area of 62.7 Å(2) at 25 °C. MD simulations with POPS lipid area constrained at different values were also performed using all-atom and aliphatic united-atom models. The optimal simulated bilayer was obtained using a model-free comparison approach. Examination of the simulated bilayer, which agrees best with the experimental scattering data, reveals a preferential interaction between Na(+) ions and the terminal serine and phosphate moieties. Long-range inter-lipid interactions were identified, primarily between the positively charged ammonium, and the negatively charged carboxylic and phosphate oxygens. The area compressibility modulus KA of the POPS bilayer was derived by quantifying lipid area as a function of surface tension from area-constrained MD simulations. It was found that POPS bilayers possess a much larger KA than that of neutral phosphatidylcholine lipid bilayers. We propose that the unique molecular features of POPS bilayers may play an important role in certain physiological functions.
Molecular imaging enables the non-invasive investigation of cellular and molecular processes. Although there are challenges to overcome, the development of targeted contrast agents to increase the sensitivity of molecular imaging techniques is essential for their clinical translation. In this study, spontaneously forming, small unilamellar vesicles (sULVs) (30 nm diameter) were used as a platform to build a bimodal (i.e., optical and magnetic resonance imaging (MRI)) targeted contrast agent for the molecular imaging of brain tumors. sULVs were loaded with a gadolinium (Gd) chelated lipid (Gd-DPTA-BOA), functionalized with targeting antibodies (anti-EGFR monoclonal and anti-IGFBP7 single domain), and incorporated a near infrared dye (Cy5.5). The resultant sULVs were characterized in vitro using small angle neutron scattering (SANS), phantom MRI and dynamic light scattering (DLS). Antibody targeted and nontargeted Gd loaded sULVs labeled with Cy5.5 were assessed in vivo in a brain tumor model in mice using time domain optical imaging and MRI. The results demonstrated that a spontaneously forming, nanosized ULVs loaded with a high payload of Gd can selectively target and image, using MR and optical imaging, brain tumor vessels when functionalized with anti-IGFBP7 single domain antibodies. The unique features of these targeted sULVs make them promising molecular MRI contrast agents.
The movement of lipids within and between intracellular membranes is mediated by different lipid transport mechanisms and is crucial for maintaining the identities of different cellular organelles. Non-vesicular lipid transport has a crucial role in intracellular lipid trafficking and distribution, but its underlying mechanisms remain unclear. Lipid-transfer proteins (LTPs), which regulate diverse lipid-mediated cellular processes and accelerate vectorial transport of lipid monomers between membranes in vitro, could potentially mediate non-vesicular intracellular lipid trafficking. Understanding the mechanisms by which lipids are transported and distributed between cellular membranes, and elucidating the role of LTPs in intracellular lipid transport and homeostasis, are currently subjects of intensive study.
RASHID, SHIRYA, JACQUES GENEST. Effect of obesity on high-density lipoprotein metabolism. Obesity. 2007;15: 2875-2888. Reduced levels of high-density lipoproteins (HDL) in non-obese and obese states are associated with increased risk for the development of coronary artery disease. Therefore, it is imperative to determine the mechanisms responsible for reduced HDL in obese states and, conversely, to examine therapies aimed at increasing HDL levels in these individuals. This paper examines the multiple causes for reduced HDL in obese states and the effect of exercise and diet-two non-pharmacologic therapies-on HDL metabolism in humans. In general, the concentration of HDL-cholesterol is adversely altered in obesity, with HDL-cholesterol levels associated with both the degree and distribution of obesity. More specifically, intra-abdominal visceral fat deposition is an important negative correlate of HDL-cholesterol. The specific subfractions of HDL that are altered in obese states include the HDL 2 , apolipoprotein A-I, and pre-1 subfrac-tions. Decreased HDL levels in obesity have been attributed to both an enhancement in the uptake of HDL 2 by adipo-cytes and an increase in the catabolism of apolipoprotein A-I on HDL particles. In addition, there is a decrease in the conversion of the pre-1 subfraction, the initial acceptor of cholesterol from peripheral cells, to pre-2 particles. Conversely , as a means of reversing the decrease in HDL levels in obesity, sustained weight loss is an effective method. More specifically, weight loss achieved through exercise is more effective at raising HDL levels than dieting. Exercise mediates positive effects on HDL levels at least partly through changes in enzymes of HDL metabolism. Increased lipid transfer to HDL by lipoprotein lipase and reduced HDL clearance by hepatic triglyceride lipase as a result of endurance training are two important mechanisms for increases in HDL observed from exercise.
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Cell membranes perform multiple functions that may be facilitated by the lateral organization of lipids and proteins into nanoscale compartments, termed membrane rafts. Understanding the origins of raft domains and the physicochemical mechanisms that control their finite small size is important for elucidating their functional significance and manipulating their properties. However, it has proven difficult to characterize rafts in cells due to the chemical complexity of biological membranes, and various models have been advanced based on theory and experiments of representative model membranes. In one popular model, a special role in the domains existence and properties has been postulated for chain-asymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain. It was proposed that these lipids align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and thus reducing domain size. Such a unique "line-active" role for hybrid lipids is an appealing explanation for nanoscopic rafts, as animal cell membranes contain few symmetric low-melting lipids but an abundance of hybrid lipids. We present data from small-angle neutron scattering and fluorescence techniques demonstrating the existence of nanoscopic and modulated liquid phase domains in a mixture composed entirely of nonhybrid lipids and cholesterol. Our results are indistinguishable from those obtained previously for mixtures containing hybrid lipids, conclusively showing that hybrid lipids are not required for the formation of nanoscopic liquid domains. These findings present new challenges for current theoretical descriptions of nanodomains.
Lipid exchange/transfer has been compared for zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC) small unilamellar vesicles (SUVs) and for the same lipids on silica (SiO2) nanoparticle supported lipid bilayers (NP-SLBs) as a function of ionic strength, temperature, temperature cycling, and NP size, above the main gel-to-liquid crystal phase transition temperature, Tm, using d- and h- DMPC and DPPC. Increasing ionic strength decreases the exchange kinetics for the SUVs, but more so for the NP-SLBs, due to better packing of the lipids and increased attraction between the lipid and support. When the NP-SLBs (or SUVs) are cycled above and below Tm, the exchange rate increases compared with exchange at the same temperature without cycling, for similar total times, suggesting that defects provide sites for more facile removal and thus exchange of lipids. Defects can occur: (i) at the phase boundaries between co-existing gel and fluid phases at Tm; (ii) in bare regions of exposed SiO2 that form during NP-SLB formation due to mismatched surface areas of lipid and NPs; and (iii) during cycling as the result of changes in area of the lipids at Tm and mismatched thermal expansion coefficient between the lipids and SiO2 support. Exchange rates are faster for NP-SLBs prepared with the nominal amount of lipid required to form a NP-SLB compared with NP-SLBs that have been prepared with excess lipids to minimize SiO2 patches. Nanosystems prepared with equimolar mixtures of NP-SLBs composed of d-DMPC (d-NP-SLBDMPC) and SUVs composed of h-DMPC (h-SUVDMPC) show that the calorimetric transition of the "donor" h-SUVDMPC decreases in intensity without an initial shift in Tm, indicating that the "acceptor" d-NP-SLBDMPC can accommodate more lipids, either through further fusion or insertion of lipids into the distal monolayer. Exchange for d-/h-NP-SLBDMPC is in the order 100 nm SiO2 > 45 nm SiO2 > 5 nm SiO2.
High-density lipoprotein (HDL) intravascular metabolism is complex, and the major HDL functions are esterification of cholesterol and reverse cholesterol transport, in which cholesterol from cells is excreted in bile. HDL has also several other antiatherogenic functions: antioxidative, vasodilatatory, anti-inflammatory, antiapoptotic, anti thrombotic, and anti infectious. Low HDL cholesterol is a major risk factor for cardiovas cular disease (CVD) and high HDL cholesterol may favor the many protective abilities of HDL. However, aspects of HDL function can be independent of HDL cholesterol levels, including the efflux of cholesterol from cells to HDL. Some populations show low incidence of CVD despite their low HDL cholesterol. Lipid exchange between HDL and other lipoproteins and cells is fundamental in HDL metabolism and reverse cholesterol transport. By determining HDL composition, lipid transfers can also affect HDL functions that depend on proteins that anchor on HDL particle surface. Cholesteryl ester protein (CETP) and phospholipid transfer protein facilitate lipid transfers among lipoprotein classes, but the role of the lipid transfers and transfer proteins in atherosclerosis and other diseases is not well established. CETP has become a therapeutic target because CETP inhibitors increase HDL cholesterol, but to date the clinical trials failed to show benefits for the patients. Recently, we introduced a practical in vitro assay to evaluate the simultaneous transfer from a donor nanoemulsion to HDL of unesterified and esterified cho lesterol, phospholipids, and triglycerides. Groups of subjects at different clinical, nutritional, and training conditions were tested, and among other findings, lower transfer ratios of unesterified cholesterol to HDL were predictors of the presence of CVD.
The mechanism of chain exchange in block polymer micelles at equilibrium is investigated using time-resolved small-angle neutron scattering (TR-SANS). The binary micelles are formed from blends of two poly(styrene-b-ethylene-alt-propylene) (PS-PEP) copolymers with different PS core block lengths, only one of which is contrast-matched with the solvent, squalane, so that the monitored scattering intensity only reflects the other species. Micelles prepared with an excess of deuterated PS chains (of the visible species) and those with the equivalent protonated PS chains are blended (“postmixed”) at room temperature, where the exchange of chains is suppressed. At several elevated temperatures these samples were monitored by TR-SANS, in which mixing of isotope-labeled visible species gives systematic reduction of scattering signals with time and provides a quantitative way to characterize the micelle exchange kinetics. Within experimental error, the results for each labeled chain (i.e., longer or shorter) in the binary micelles are identical to those recently reported for the same labeled chains in the corresponding single block copolymer component micelles, thus proving that chain exchange in these micelles involves independent chain motion. This reinforces the important conclusions that the single-chain exchange mechanism dominates in the studied micelle solutions and that micelle fusion or fission events are rare.
The role of core block size dispersity on the rate of molecular exchange in spherical micelles formed from 1% by volume poly(styrene-b-ethylenepropylene) (PS-PEP) diblock copolymers in squalane (C30H62) was investigated using time-resolved small-angle neutron scattering (TR-SANS). Separate copolymer solutions (total polymer 1% by volume) containing either deuterium labeled (dPS) or normal (hPS) poly(styrene) core blocks were prepared and mixed at room temperature, below the core glass transition temperature. Each preparation (dPS or hPS) contained equal volume fractions of Mn = 26 and 42 kg/mol (h-equivalent) poly(styrene) blocks. Heating to temperatures between 87 and 146 °C resulted in block copolymer exchange as evidenced by a systematic reduction in the SANS intensity; C30H62 and C30D62 were blended so as to contrast match the fully exchanged cores. Following a protocol established in a previous report, the time-dependent intensity data were shifted with respect to time and temperature, leading to a master curve covering nearly 7 orders of magnitude in reduced time. These results are quantitatively accounted for by summing the weighted relaxation functions obtained from the individual components, consistent with a previously published model that accounts for the dramatic sensitivity of the molecular exchange dynamics to core block dispersity.
Lipid-based nanodiscs (bicelles) are able to form in mixtures of long- and short-chain lipids. Initially, they are of uniform size but grow upon dilution. Previously, nanodisc growth kinetics have been studied using time-resolved small angle neutron scattering (SANS), a technique which is not well suited for probing their change in size immediately after dilution. To address this, we have used dynamic light scattering (DLS), a technique which permits the collection of useful data in a short span of time after dilution of the system. The DLS data indicate that the negatively charged lipids in nanodiscs play a significant role in disc stability and growth. Specifically, the charged lipids are most likely drawn out from the nanodiscs into solution, thereby reducing interparticle repulsion and enabling the discs to grow. We describe a population balance model, which takes into account Coulombic interactions and adequately predicts the initial growth of nanodiscs with a single parameter - i.e., surface potential. The results presented here strongly support the notion that the disc coalescence rate strongly depends on nanoparticle charge density. The present system containing low-polydispersity lipid nanodiscs serves as a good model for understanding how charged discoidal micelles coalesce.
The self-assembling morphologies of low-concentration (mostly 1 and 10mg/mL) bicellar mixtures composed of zwitterionic dipalmitoyl (di-C16) phosphatidylcholine (DPPC), dihexanoyl (di-C6) phosphatidylcholine (DHPC), and negatively charged dipalmitoyl (di-C16) phosphatidylglycerol (DPPG) were investigated using small angle neutron scattering, dynamic light scattering and transmission electron microscopy. A polyethylene glycol conjugated (PEGylated) lipid, distearoyl phosphoethanolamine-[methoxy (polyethyleneglycol)-2000] (PEG2000-DSPE), was incorporated in the system at 5 mole% of the total lipid composition. The effects of several parameters on the spontaneous structures are studied, including temperature, lipid concentration, salinity, and PEG2000-DSPE. In general, nanodiscs (bicelles) were observed at low temperatures (below the melting temperature, TM of DPPC) depending on the salinity of the solutions. Nanodisc-to-vesicle transition is found upon the elevation of temperature (above TM) in the cases of low lipid concentration in absence of PEG2000-DSPE or high salinity. Both addition of PEG2000-DSPE and high lipid concentration stabilize the nanodiscs, preventing the formation of multilamellar vesicles, while high salinity promotes vesiculation and the formation of aggregation. This study suggests that the stability of such nanodiscs is presumably controlled by the electrostatic interactions, the steric effect induced by PEG2000-DSPE and the amount of DHPC located at the disc rim.
Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of β-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and β-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer's disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where β-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36.
The family of StAR related lipid transfer proteins (START) is so-named based on the distinctive capacity for these proteins to transport lipids between membranes. The START domain is a module of about 210 residues, which binds lipids such as glycerolipids, sphingolipids and sterols. This domain has a deep lipid-binding pocket - that shields the hydrophic ligand from the external aqueous environment - covered by a lid. Based on their homology, the fifteen START proteins in mammals have been allocated to six distinct subfamilies, each subfamily being more specialized in the transport and/or sensing of a lipid ligand species. However within the same subgroup, their expression profile and their subcellular localization distinguish them and are critical for their different biological functions. Indeed, START proteins act in a variety of distinct physiological processes, such as lipid transfer between intracellular compartments, lipid metabolism and modulation of signaling events. Mutation or deregulated expression of START proteins is linked to pathological processes, including genetic disorders, autoimmune diseases and cancers. Besides the common single START domain, which is always located at the carboxy-terminal end in mammals, most START proteins harbor additional domains predicted to be critical in favoring lipid exchange. Evidence from well characterized START proteins indicates that these additional domains might be tethering machineries able to bring distinct organelles together and create membrane contact sites prone to lipid exchange via the START domain.
Nanometer-scale domains in cholesterol-rich model membranes emulate lipid rafts in cell plasma membranes (PMs). The physicochemical mechanisms that maintain a finite, small domain size are, however, not well understood. A special role has been postulated for chain-asymmetric or hybrid lipids, having a saturated sn-1 chain and an unsaturated sn-2 chain. Hybrid lipids generate nanodomains in some model membranes, and are also abundant in the PM. It was proposed that they align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and thus reducing domain size. We used small-angle neutron scattering and fluorescence techniques to detect nanoscopic and modulated liquid phase domains in a mixture composed entirely of non-hybrid lipids and cholesterol. Our results are indistinguishable from those obtained previously for mixtures containing hybrid lipids, conclusively showing that hybrid lipids are not required for the formation of nanoscopic liquid domains, and strongly implying a common mechanism for the overall control of raft size and morphology. We discuss implications of these findings for theoretical descriptions of nanodomains.
In need for nonperturbing but spectroscopically unique environments for studies of lipid/lipid and lipid/protein interactions, we have investigated the thermodynamic properties of deuterated and undeuterated dipalmitoyl phosphatidylcholines as well as the mixtures of both. The data have been collected using the highly sensitive differential scanning calorimetry. From the results of these measurements we conclude that mixtures of deuterated and undeuterated lipids behave ideally. The transition enthalpies, transition entropies and the cooperative unit are given as a function of the input ratio of the components and the phase diagram is constructed, showing that the deuterated lipids are suitable as nonperturbing probes.
Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease.
Synthetic lipids with a nitroxide or a fluorescent probe have been extensively used during the last 30 years to determine the transmembrane diffusion of phospholipids in artificial or biological membranes. However, the relevance of data obtained with these modified lipids has sometimes been questioned. Beside possible artefacts introduced by the reporter probe, synthetic lipids used in cells often contain a short fatty acid chain in the sn-2 position, which gives them higher water solubility than naturally occurring lipids. In the present review, we have attempted to give a critical appraisal. Main strategies are recalled and important discoveries obtained with lipid probes on transmembrane lipid traffic in eukaryotic cells are briefly summarized. Examples of artefacts caused by lipid probes are given. Comparisons between data obtained by different techniques such as ESR and fluorescence allow us to emphasize the complementary character of the two approaches and more generally show the necessity to use several probes before drawing conclusions concerning endogenous lipids. In spite of these pitfalls, overall, lipid probes have provided a wealth of useful information that, to date, cannot be obtained with unlabeled lipids.
Both solution and solid state nuclear magnetic resonance (NMR) techniques for structural determination are advancing rapidly such that it is possible to contemplate bringing these techniques to bear upon integral membrane proteins having multiple transmembrane segments. This review outlines existing and emerging options for model membrane media for use in such studies and surveys the special considerations which must be taken into account when preparing larger membrane proteins for NMR spectroscopic studies.
The physical stability of the four liposomal systems was determined on storage at 4 and 25°C over a 6-month period. A correlation of the mean volume diameter, zeta potential and pH lead to the conclusion that stability follows the order of egg lecithin (PC)/cholesterol (CH)/stearylamine (SA) < PC/CH/phosphatidylserine (PS) < bovine brain ceramides (CM)/CH/palmitic acid (PA)/CS < PC/CH/cholesteryl sulphate (CS) at 4°C, as well as at 25°C, after a 6-month storage period. Large unilamellar vesicles (REV) proved to be superior to multilamellar liposomes (MLV) and dehydration/rehydration liposomes (DRV) systems as far as physical stability was concerned. Instability was exaggerated in the systems stored at 25°C as compared to storage at 4°C.
In recent years many articles have been devoted to the study of mixtures of labeled and unlabeled polymers by small-angle neutron scattering. The main purpose of these articles was to extract from the measured scattered intensity the structure factor of the macromolecules in order to obtain information on their conformation. This formulation has been limited to systems with a small number of constituents and often assumes incompressibility. The authors generalize these treatments to systems having an arbitrary number of constituents without making the assumption of incompressibility at the outset.
The observation of lateral phase separation in lipid bilayers has received considerable attention, especially in connection to lipid raft phenomena in cells. It is widely accepted that rafts play a central role in cellular processes, notably signal transduction. While micron-size domains are observed with some model membrane mixtures, rafts much smaller than 100 nm-beyond the reach of optical microscopy-are now thought to exist, both in vitro and in vivo. We have used small-angle neutron scattering (SANS), a probe free technique, to measure the size of nanoscopic membrane domains in unilamellar vesicles with unprecedented accuracy. These experiments were performed using a four-component model system containing fixed proportions of cholesterol and the saturated phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), mixed with varying amounts of the unsaturated phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We found that liquid domain size increases with the extent of acyl chain unsaturation (DOPC:POPC ratio). Furthermore, we find a direct correlation between domain size and the mismatch in bilayer thickness of the coexisting liquid-ordered and liquid-disordered phases, suggesting a dominant role for line tension in controlling domain size. While this result is expected from line tension theories, we provide the first experimental verification in free-standing bilayers. Importantly, we also find that changes in bilayer thickness, which accompany changes in the degree of lipid chain unsaturation, are entirely confined to the disordered phase. Together, these results suggest how the size of functional domains in homeothermic cells may be regulated through changes in lipid composition.
Molecular exchange kinetics of diblock copolymers forming spherical micelles packed on a body-centered cubic (bcc) lattice were investigated using time-resolved small-angle neutron scattering (TR-SANS). Ordered arrays of polystyrene spheres were prepared by mixing 15 vol % poly(styrene-b-ethylene-alt-propylene) (PS−PEP) in squalane, a highly selective solvent for the PEP block. Two pairs of diblock copolymers were examined, characterized by two different PS core block molecular weights (MPS = 26500 and 42800). Each pair contained two nearly identical diblock copolymers, one with a deuterated PS block (dPS−PEP) and the other with a protonated PS block (hPS−PEP). Protonated and deuterated squalane were blended to achieve a contrast-matched condition with uniformly mixed dPS and hPS (50/50 by volume) core chains. Beginning with a statistically random array of pure dPS and hPS cores distributed on the bcc lattice, molecular exchange was monitored at multiple temperatures by TR-SANS. Exchange of deuterated and protonated chains results in a decay in SANS intensity, which yields a kinetic function revealing a broad spectrum of relaxation times. These results are qualitatively consistent with our previously reported findings for molecular exchange between PS−PEP micelles in a dilute (1 vol %) squalane solution. However, the measured characteristic time constant for the concentrated, ordered system is more than an order of magnitude slower than in the dilute, disordered dispersion.
Bilayered mixed micelles or bicelles are magnetically anisotropic, self-assembling model membrane structures comprised of long-chain phospholipids and short-chain detergent molecules. In the most widely accepted model of this system, the bicelle is discoid in shape, with the short-chain DHPC molecules aggregating to form rims around long-chain DMPC bilayers. While this model is consistent with most NMR and scattering data (X-ray and neutron), it inadequately describes the liquid-crystalline behavior of bicelle solutions at temperatures where magnetic alignment occurs. Temperature plays a central role in the structure of lipid aggregates, and the impact of temperature on bicelles has not been studied as extensively as composition and concentration. Therefore, a series of fluorescence probe and resonance energy transfer (FRET) measurements of labeled bicelle solutions as a function of temperature were conducted to monitor lipid mixing as an indication of bicelle structure and aggregation. The results of these measurements are not consistent with the large-scale changes in lipid mixing with temperature that have been attributed to bicelle solutions in other studies. The spectral data indicate that there is reorganization within mixed lipid aggregates as a function of temperature and bilayer fusion. In an attempt to reconcile these data with physical data and the theory of liquid-crystalline behavior, the authors speculate that the structure of bicelles is an interconnected network of DMPC bilayers interrupted by DHPC rimmed pores at elevated temperatures.
A systematic study of the structural determinants involved in the spontaneous transfer of molecules between single bilayer vesicles of phosphatidylcholine is reported. All of the molecules studied contain a pyrenyl moiety whose excimer fluorescence provides a direct measure of the changes in its microscopic concentration. These compounds include pyrenyl alkanes, alcohols, carboxylic acids, and their methyl esters. In each group, transfer between vesicles occurs via the intervening aqueous phase. The rate of transfer is a function of both the hydrophobicity (chain length) and the hydrophilicity (polar or nonpolar) of the transferred species. The rates of transfer can be expressed in terms of a free energy of activation, ΔG‡, which is calculated from absolute rate theory. A good correlation exists between ΔG‡ and ΔGt, the free energy of molecular transfer from a hydrophobic environment to the aqueous phase. The rate of transfer increased both with decreasing chain length in a given homologous series and with the polarity of the substituents if the number of methylene units is constant. The incremental ΔG‡ for the polar compounds was ≃740 cal/methylene unit, whereas the corresponding value for the alkyl pyrenes is ∼900 cal/methylene unit. These values are similar to the reported ΔGt per methylene unit calculated from equilibrium measurements. The ΔG‡ per methylene unit of the polar compounds reflected changes in the ΔH‡ since ΔS‡ was independent of chain length. By contrast, the alkyl pyrenes exhibited very large changes in ΔG‡ with increasing chain length (≃2 kcal/methylene unit) that are, in part, compensated by changes in ΔS‡. As a consequence, only a small difference in the contribution of each methylene to ΔG‡ of transfer of alkanes and amphiphiles is predicted.
The transfer of L(α) phosphatidylcholines of different acyl chain length between small unilamellar vesicles via the aqueous phase was studied by using high-sensitivity differential scanning calorimetry (hs-DSC), 1H NMR, dynamic light scattering, and freeze-fracture electron microscopy. Two cases of lipid transfer were studied: (1) the asymmetric lipid transfer between phosphatidylcholine vesicles with different acyl chain lengths of the lipids; (2) the symmetric lipid transfer between phosphatidylcholine vesicles of the same type of lipid by using acyl chain deuteriated and protonated lipid analogues. From the hs-DSC data the off rates of the lipids were calculated for both types of transfer as a function of incubation temperature of the vesicles and the acyl chain lengths of the lipids by using a kinetic model. Significant differences between the two types of lipid transfer were found. In the asymmetric case a net transfer of lipids from the vesicle fraction with the lower lipid chain melting temperature (donor vesicles) to those with the higher one (acceptor vesicles) was observed even while the latter were in the gel state. Additionally, the transfer kinetics was strongly dependent on the proportion between donor and acceptor vesicles. In the symmetric case the lipid transfer between the two vesicle populations was nearly equal (i.e., lipid transfer in a 1:1 ratio between the vesicles) and occurred only if all lipids were in the liquid-crystalline state. A further characteristic of the asymmetric lipid transfer was an acceleration of the flip-flop rate of the acceptor vesicles by at least 1 order of magnitude in the initial stage of transfer. The dependence of the transfer kinetics on the incubation temperature, the acyl chain lengths of the lipids, and the total lipid concentration was found to be similar for both types of lipid transfer, provided that all lipids were in the fluid state. It is further shown that lipid transfer is a characteristic of small sonicated vesicles, whereas larger vesicles prepared by detergent dialysis exchanged lipids mainly by vesicle fusion.
Bicelles are an attractive membrane mimetic system because of their planar surface and lipid composition, which resemble biological membranes. In addition, their orientation and morphologic properties make them amenable to solid- and solution-state NMR. This article reviews the physical properties of bicelles, such as magnetic alignment and viscosity as well as the different models proposed in the literature to explain the bicelle morphology. The utility of bicelles for studying the interaction and structure of membrane peptides and proteins by solid- and solution-state NMR is also presented, along with the advantages and limitations of bicelles. © 2005 Wiley Periodicals, Inc. Concepts Magn Reson Part A 24A: 17–37, 2005.
An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed.
Negatively charged phospholipids are an important component of biological membranes. The thermodynamic parameters governing self-assembly of anionic phospholipids are deduced here from isothermal titration calorimetry. Heats of demicellization were determined for dioctanoyl phosphatidylglycerol (PG) and phosphatidylserine (PS) at different ionic strengths, and for dioctanoyl phosphatidic acid at different pH values. The large heat capacity (ΔC°(P) ∼ -400 J.mol(-1) K(-1) for PG and PS), and zero enthalpy at a characteristic temperature near the physiological range (T(∗) ~ 300 K for PG and PS), demonstrate that the driving force for self-assembly is the hydrophobic effect. The pH and ionic-strength dependences indicate that the principal electrostatic contribution to self-assembly comes from the entropy associated with the electrostatic double layer, in agreement with theoretical predictions. These measurements help define the thermodynamic effects of anionic lipids on biomembrane stability.
Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., [DMPG]/[DMPC] = 0.01 and 0.001) and two solution salinities (i.e., [NaCl] = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 °C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 °C), but transform into uniform size, nanoscopic ULVs after incubation at 10 °C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 °C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 °C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical applications that depend on monodisperse lipid-based systems for therapeutic and diagnostic purposes.
The structural parameters of fluid phase bilayers composed of phosphatidylcholines with fully saturated, mixed, and branched fatty acid chains, at several temperatures, have been determined by simultaneously analyzing small-angle neutron and X-ray scattering data. Bilayer parameters, such as area per lipid and overall bilayer thickness have been obtained in conjunction with intrabilayer structural parameters (e.g. hydrocarbon region thickness). The results have allowed us to assess the effect of temperature and hydrocarbon chain composition on bilayer structure. For example, we found that for all lipids there is, not surprisingly, an increase in fatty acid chain trans-gauche isomerization with increasing temperature. Moreover, this increase in trans-gauche isomerization scales with fatty acid chain length in mixed chain lipids. However, in the case of lipids with saturated fatty acid chains, trans-gauche isomerization is increasingly tempered by attractive chain-chain van der Waals interactions with increasing chain length. Finally, our results confirm a strong dependence of lipid chain dynamics as a function of double bond position along fatty acid chains.
Understanding the interaction between functional nanoparticles and cell membranes is critical to use nanomaterials for broad biomedical applications with minimal cytotoxicity. In this work, we have investigated the effect of adsorbed semihydrophobic nanoparticles (NPs) on the dynamics and morphology of model cell membranes. We have systematically varied the degree of surface hydrophobicity of carboxyl end-functionalized polystyrene NPs of varied size in buffer solutions with varied ionic strength. It is observed that semihydrophobic NPs can readily adsorb on neutral SLBs and drag lipids from SLBs to NP surfaces. Above a critical NP concentration, the disruption of SLBs is observed, accompanied with the formation and rapid growth of lipid-poor regions on NP-adsorbed SLBs. In the study of the effect of solution ionic strength on NP surface hydrophobic degree and the growth of lipid-poor regions, we have concluded that the hydrophobic interaction enhanced by screened electrostatic interaction underlies the envelopment of NPs by lipids that are attracted from SLBs to the surface of NPs or their aggregates. Hence, the formation and growth of lipid-poor regions, or vaguely referred as "pores" or "holes" in the literature, can be controlled by NP concentration, size, and surface hydrophobicity, which is critical to design functional nanomaterials for effective nanomedicine while minimizing possible cytotoxicity.
Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.
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Abstract: The structural phases of magnetically alignable lipid mixtures were investigated as a function of temperature and lipid concentration using small-angle neutron scattering (SANS). Two systems were examined: (a) an aqueous mixture of DMPC (dimyristoyl phosphatidylcholine) and DHPC (dihexanoyl phosphatidylcholine) lipids doped with Tm3+ ions resulting in the positive alignment of the system with the applied magnetic field and (b) the above aqueous Tm3+ doped lipid mixture containing a negatively charged lipid, DMPG (dimyristoylphosphatidylglycerol). For both systems, three different scattering patterns were observed corresponding to distinct structural phases at specific temperatures and lipid concentrations. At 45 C and a lipid concentration of >0.05 g/mL, the high-viscosity liquid crystalline phase was found to be a perforated and possibly undulating lamellar phase consistent with NMR results. Upon dilution (<0.05 g/mL) at the same temperature (45 C), the perforated lamellar phase transformed into a unilamellar vesicular phase, in which the bilayers may also be perforated. Below about 25 C, the viscosity decreases considerably and the scattering data suggest that the lamellae present at higher temperatures break up into smaller entities characterized by the bicellar morphology proposed previously for the nondoped system. The structural dimensions of the vesicular and bicellar phases have been determined as a function of lipid concentrations from the SANS data. In the lamellar phase, the influence of Tm3+ ions and DMPG on bilayer structure (e.g., lamellar repeat spacing, bilayer rigidity, and magnetic alignment) were also investigated.
Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca(2+) homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.
Nanodiscs are phospholipid-protein complexes which are relevant to nascent high-density lipoprotein and are applicable as a drug carrier and a tool to immobilize membrane proteins. We evaluated the structure and dynamics of the nanoparticles consisting of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (apoA-I) with small-angle neutron scattering (SANS) and fluorescence methods and compared them with static/dynamic properties for large unilamellar vesicles. SANS revealed that the nanodisc includes a lipid bilayer with a thickness of 44 A and a radius of 37 A, in which each lipid occupies a smaller area than the reported molecular area of DMPC in vesicles. Fluorescence measurements suggested that DMPC possesses a lower entropy in nanodiscs than in vesicles, because apoA-I molecules, which surround the bilayer, force closer lipid packing, but allow water penetration to the acyl chain ends. Time-resolved SANS experiments revealed that nanodiscs represent a 20-fold higher lipid transfer via an entropically favorable process. The results put forward a conjunction of static/dynamic properties of nanodiscs, where the entropic constraints are responsible for the accelerated desorption of lipids.
In this study, the center-of-mass diffusion and shape fluctuations of large unilamellar 1-palmitoyl-2-oleyl-sn-glycero-phosphatidylcholine vesicles prepared by extrusion are studied by means of neutron spin echo in combination with dynamic light scattering. The intermediate scattering functions were measured for several different values of the momentum transfer, q, and for different cholesterol contents in the membrane. The combined analysis of neutron spin echo and dynamic light scattering data allows calculation of the bending elastic constant, kappa, of the vesicle bilayer. A stiffening effect monitored as an increase of kappa with increasing cholesterol molar ratio is demonstrated by these measurements.
Interfacial tensions of egg yolk phosphatidylcholine (PC) and cholesterol monolayers adsorbed at the triolein-saline interface were measured in the presence and absence of pig apolipoprotein A-1 (apoA-1) in the saline phase. In the absence of apoA-1, the adsorptions of PC and cholesterol at the interface from the triolein phase are cooperative, showing large lateral attractive interactions between the PC molecules and the cholesterol molecules in the monolayer. In the presence of apoA-1, the PC adsorption is anti-cooperative, indicating strong lateral attractive interactions between the PC and the apoA-1 molecules, i.e., apparently, repulsive lateral interactions between the PC molecules. On the other hand, lateral interactions of very low magnitude are observed between the cholesterol and apoA-1 molecules in the monolayer. Values of the lateral interaction energy are evaluated from the adsorption data by the Defay-Prigogine-Flory theory of monolayers. The large difference in lateral interaction energy with apoA-1 between PC and cholesterol in a mixed monolayer is discussed in connection with current problems in lipoprotein catabolism: reverse cholesterol transport, alterations in affinity of lipid particles to apoA-1, and formation of high-density lipoproteins and abnormal lipoproteins.
The rates of spontaneous interbilayer and transbilayer exchange of [3H]dimyristoylphosphatidylcholine ([3H]DMPC) were examined in DMPC and DMPC/dimyristoylphosphatidylethanolamine (DMPE) large unilamellar vesicles in the liquid-crystalline-, gel-, and mixed-phase states. DMPC desorption rates from either gel or liquid-crystalline phases containing DMPE are very similar to the corresponding rates from pure DMPC gel or liquid-crystalline phases. This is not the case for DMPC desorption from distearoylphosphatidylcholine (DSPC)-containing gel phases, where the desorption rates are significantly faster than from a pure DMPC gel phase [Wimley, W. C., & Thompson, T. E. (1990) Biochemistry 29, 1296-1303]. We proposed that the DMPC/DSPC behavior results from packing defects in gel phases composed of both DMPC and DSPC molecules because of the four-carbon difference in the acyl chain lengths of the two species. The present results strongly support this hypothesis because no such anomalous behavior is observed in DMPC/DMPE, which is similar to DMPC/DSPC in phase behavior but does not have the chain length difference. The inclusion of 10-30 mol % DMPE in DMPC bilayers was also found to have a significant effect on the rate of transbilayer movement (flip-flop) of [3H]DMPC in the liquid-crystalline phase. Between 10 and 30 mol % DMPE, flip-flop of DMPC is slowed by at least 10-fold relative to flip-flop in DMPC bilayers, and the entropy and enthalpy of flip-flop activation are both substantially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. These vaccines are weakly or non-immunogenic and cannot, therefore, be used effectively in the absence of immunological adjuvants (agents that can induce strong immunity to antigens). Owing to the toxicity of adjuvants, only one (aluminium salts) has hitherto been licensed for use in humans, and it is far from ideal. In this article, Gregory Gregoriadis discusses the use of liposomes as an alternative safe, versatile, universal adjuvant that can induce humoral- and cell-mediated immunity to antigens when administered parenterally or enterally.
The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Micelles formed from sodium glycocholate and dimyristoylphosphatidylcholine are demonstrated to form a magnetic field orientable liquid crystal within narrow ranges of composition and temperature. The utility of this medium in structural investigations of biological membrane components using deuterium NMR is discussed.
A systemic formalism is developed that shows how the results for absolute specific volumes of multilamellar lipid dispersions may be combined with results from diffraction studies to obtain quantitative characterizations of the average structure of fully hydrated lipid bilayers. Quantities obtained are the area per molecule, the thickness and volumes of the bilayer, the water layer, the hydrocarbon chain layer and the headgroup layer, and where appropriate, the tilt angle of the hydrocarbon chains. In the case of the C phase of DPPC this formalism leads to the detection of inconsistencies between three data. Results for the G phases of DPPC and DLPE are in reasonable agreement with, though more comprehensive than, previous work that used fewer data and equations. Various diffraction data for the F phase of DPPC are in disagreement and it is shown how this disagreement affects results for the bilayer structure. A recent method of McIntosh and Simon for obtaining fluid phase structure utilizing gel phase structure is slightly modified to obtain results for the F phase of DLPE. Methods of obtaining the average methylene and methyl volumes in the fluid phases are critically examined.