Anesthetic drugs have been in use for over 160 years in surgery, but their mode of action remains largely unresolved. We have studied the effect of (R)-(-)-ketamine on the biophysical properties of ...lipid model membranes composed of palmitoyloleoylphosphatidylcholine by a combination of X-ray diffraction and all-atom molecular dynamics simulations. In agreement with several previous studies, we do not find significant changes to the membrane thickness and lateral area per lipid up to 8 mol % ketamine content. However, we observed that the insertion of ketamine within the lipid/water interface caused significant changes of lateral pressure and a pressure shift toward the center of the bilayer. The changes are predicted to be large enough to affect the opening probability of ion channels as derived for two protein models. Depending on the protein model, we found inhibition values of IC(50) = 2 mol % and 18 mol % ketamine, corresponding to approximately 0.08 and 0.9 muM concentrations in the blood circulation, respectively. This compares remarkably well with clinical applied concentrations. We thus provide evidence for a lateral pressure mediated mode of anesthesia, first proposed more than 10 years ago.
Membrane-active molecules provide a promising strategy to target and kill pathogenic bacteria. Understanding how specific molecular features drive interactions with membrane components and ...subsequently cause disruption that leads to antimicrobial activity is a crucial step in designing next-generation treatments. Here, we test a library of lipid-like compounds (lipidoids) against Gram-negative bacteria Escherichia coli to garner in-depth structure–activity relationships using antimicrobial assays. Modular lipidoid molecules were synthesized in high-throughput, such that we could analyze 104 compounds with variable combinations of hydrophobic tails and cationic headgroups. Antibacterial activity was strongly correlated to specific structural features, including tail hydrophobicity and headgroup charge density, and also to the overall molecular shape and propensity for self-assembly into curved liquid crystalline phases. Dye permeabilization assays showed that E. coli membranes were permeabilized by lipidoids, confirming their membrane-active nature. The reduced permeabilization, as compared to Gram-positive Bacillus subtilis, alludes to the challenge of permeabilizing the additional outer membrane layer of E. coli. The effect of headgroup solubility in gemini-type lipidoids was also demonstrated, revealing that a headgroup with a more hydrophilic spacer between amine groups had enhanced activity against B. subtilis but not E. coli. This provides insight into features enabling outer membrane penetration and governing selectivity between bacterial species.
We studied the transleaflet coupling of compositionally asymmetric liposomes in the fluid phase. The vesicles were produced by cyclodextrin-mediated lipid exchange and contained dipalmitoyl ...phosphatidylcholine (DPPC) in the inner leaflet and different mixed-chain phosphatidylcholines (PCs) as well as milk sphingomyelin (MSM) in the outer leaflet. In order to jointly analyze the obtained small-angle neutron and X-ray scattering data, we adapted existing models of trans-bilayer structures to measure the overlap of the hydrocarbon chain termini by exploiting the contrast of the terminal methyl ends in X-ray scattering. In all studied systems, the bilayer-asymmetry has large effects on the lipid packing density. Fully saturated mixed-chain PCs interdigitate into the DPPC-containing leaflet and evoke disorder in one or both leaflets. The long saturated acyl chains of MSM penetrate even deeper into the opposing leaflet, which in turn has an ordering effect on the whole bilayer. These results are qualitatively understood in terms of a balance of entropic repulsion of fluctuating hydrocarbon chain termini and van der Waals forces, which is modulated by the interdigitation depth. Monounsaturated PCs in the outer leaflet also induce disorder in DPPC despite vestigial or even absent interdigitation. Instead, the transleaflet coupling appears to emerge here from a matching of the inner leaflet lipids to the larger lateral lipid area of the outer leaflet lipids.
Graphical abstract
All plasma membranes exhibit asymmetry, both in lipid distribution and protein topology. Cells invest significant free energy to uphold this asymmetry.Despite its critical physiological role, our ...quantitative understanding of membrane asymmetry is limited, due primarily to insufficient in vitro methods for creating asymmetric model membranes.New experimental protocols now allow for the preparation of vesicles with tailored, asymmetric lipid distributions.Leaflets with distinct lipid compositions can affect each other’s properties, such as lateral diffusion, hydrocarbon chain ordering, and bending elasticity.Integral membrane proteins not only adapt to lipid asymmetry but also exhibit asymmetric topological orientations, which in turn can depend on lipid composition.
Plasma membranes utilize free energy to maintain highly asymmetric, non-equilibrium distributions of lipids and proteins between their two leaflets. In this review we discuss recent progress in quantitative research enabled by using compositionally controlled asymmetric model membranes. Both experimental and computational studies have shed light on the nuanced mechanisms that govern the structural and dynamic coupling between compositionally distinct bilayer leaflets. This coupling can increase the membrane bending rigidity and induce order – or lipid domains – across the membrane. Furthermore, emerging evidence indicates that integral membrane proteins not only respond to asymmetric lipid distributions but also exhibit intriguing asymmetric properties themselves. We propose strategies to advance experimental research, aiming for a deeper, quantitative understanding of membrane asymmetry, which carries profound implications for cellular physiology.
Plasma membranes utilize free energy to maintain highly asymmetric, non-equilibrium distributions of lipids and proteins between their two leaflets. In this review we discuss recent progress in quantitative research enabled by using compositionally controlled asymmetric model membranes. Both experimental and computational studies have shed light on the nuanced mechanisms that govern the structural and dynamic coupling between compositionally distinct bilayer leaflets. This coupling can increase the membrane bending rigidity and induce order – or lipid domains – across the membrane. Furthermore, emerging evidence indicates that integral membrane proteins not only respond to asymmetric lipid distributions but also exhibit intriguing asymmetric properties themselves. We propose strategies to advance experimental research, aiming for a deeper, quantitative understanding of membrane asymmetry, which carries profound implications for cellular physiology.
Bicontinuous cubic phases offer advantageous routes to a broad range of applied materials ranging from drug delivery devices to membranes. However, a priori design of molecules that assemble into ...these phases remains a technological challenge. In this article, a high‐throughput synthesis of lipidoids that undergo protonation‐driven self‐assembly (PrSA) into liquid crystalline (LC) phases is conducted. With this screening approach, 12 different multi‐tail lipidoid structures capable of assembling into the bicontinuous double gyroid phase are discovered. The large volume of small‐angle X‐ray scattering (SAXS) data uncovers unexpected design criteria that enable phase selection as a function of lipidoid headgroup size and architecture, tail length and architecture, and counterion identity. Surprisingly, combining branched headgroups with bulky tails forces lipidoids to adopt unconventional pseudo‐disc conformations that pack into double gyroid networks, entirely distinct from other synthetic or biological amphiphiles within bicontinuous cubic phases. From a multitude of possible applications, two examples of functional materials from lipidoid liquid crystals are demonstrated. First, the fabrication of gyroid nanostructured films by interfacial PrSA, which are rapidly responsive to the external medium. Second, it is shown that colloidally‐dispersed lipidoid cubosomes, for example, for drug delivery, are easily assembled using top‐down solvent evaporation methods.
Screening a library of 96 multi‐tail lipidoids for protonation‐driven self‐assembly reveals a number of molecules that assemble into the bicontinuous cubic gyroid phase. Unlike lipids and other synthetic amphiphiles, lipidoids appear to adopt unusual disc‐like conformations that stack into the 3D network structures. These phases can be fabricated into films and colloidally‐stable particles for a wide range of potential applications.
generation of red blood cells (cRBCs) is an attractive tool in basic research and for replacing blood components donated by volunteers. As a prerequisite for the survival of cRBCs during storage as ...well as in the circulation, the quality of the membrane is of utmost importance. Besides the cytoskeleton and embedded proteins, the lipid bilayer is critical for membrane integrity. Although cRBCs suffer from increased fragility, studies investigating the lipid content of their membrane are still lacking. We investigated the membrane lipid profile of cRBCs from CD34
human stem and progenitor cells compared to native red blood cells (nRBCs) and native reticulocytes (nRETs).
erythropoiesis was performed in a well-established liquid assay. cRBCs showed a maturation grade between nRETs and nRBCs. High-resolution mass spectrometry analysis for cholesterol and the major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and lysophosphatidylcholin, demonstrated severe cholesterol deficiency in cRBCs. Although cRBCs showed normal deformability capacity, they suffered from increased hemolysis due to minimal changes in the osmotic conditions. After additional lipid supplementation, especially cholesterol during culturing, the cholesterol content of cRBCs increased to a subnormal amount. Concurrently, the osmotic resistance recovered completely and became comparable to that of nRETs. Minor differences in the amount of phospholipids in cRBCs compared to native cells could mainly be attributed to the ongoing membrane remodeling process from the reticulocyte to the erythrocyte stage. Obtained results demonstrate severe cholesterol deficiency as a reason for enhanced fragility of cRBCs. Therefore, the supplementation of lipids, especially cholesterol during
erythropoiesis may overcome this limitation and strengthens the survival of cRBCs
and
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We report the real-time response of
to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved ...synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s-much faster than previously considered. Final intracellular AMP concentrations of ∼80-100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.
The highly successful scattering density profile (SDP) model, used to jointly analyze small‐angle X‐ray and neutron scattering data from unilamellar vesicles, has been adapted for use with data from ...fully hydrated, liquid crystalline multilamellar vesicles (MLVs). Using a genetic algorithm, this new method is capable of providing high‐resolution structural information, as well as determining bilayer elastic bending fluctuations from standalone X‐ray data. Structural parameters such as bilayer thickness and area per lipid were determined for a series of saturated and unsaturated lipids, as well as binary mixtures with cholesterol. The results are in good agreement with previously reported SDP data, which used both neutron and X‐ray data. The inclusion of deuterated and non‐deuterated MLV neutron data in the analysis improved the lipid backbone information but did not improve, within experimental error, the structural data regarding bilayer thickness and area per lipid.
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