Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure–property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach—combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations—we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer’s packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure–property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol’s role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid–protein interactions.

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization.

The pH-low insertion peptide (pHLIP) is a leading peptide technology to target the extracellular acidosis that characterizes solid tumors. The pHLIP binds to lipid membranes, and responds to acidification by undergoing a coupled folding/membrane insertion process. In the final transmembrane state, the C terminus of pHLIP gets exposed to the cytoplasm of the target cell, providing a means to translocate membrane-impermeable drug cargoes across the plasma membrane of cancer cells. There exists a need to develop improved pHLIP variants to target tumors with greater efficiency. Characterization of such variants typically relies on determining the pK parameter, the pH midpoint of peptide insertion into the lipid bilayer. Here we report that the value of the pK can be strongly dependent on the method used for its determination. Membrane insertion of pHLIP involves at least four intermediate states, which are believed to be linked to the staggered titration of key acidic residues. We propose that some spectroscopic methods are influenced more heavily by specific membrane folding intermediates, and as a result yield different pK values. To address this potential problem, we have devised an assay to independently monitor the environment of the two termini of pHLIP. This approach provides insights into the conformation pHLIP adopts immediately before the establishment of the transmembrane configuration. Additionally, our data indicate that the membrane translocation of the C terminus of pHLIP, the folding step more directly relevant to drug delivery, occurs at more acidic pH values than previously considered. Consequently, such a pK difference could have substantial ramifications for assessing the translocation of drug cargoes conjugated to pHLIP.

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods-small angle X-ray and neutron scattering and cryogenic electron microscopy-we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only $35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.

The pH-low insertion peptide (pHLIP) targets acidic diseases such as cancer. The acidity of the environment causes key aspartic acids in pHLIP to become protonated, causing the peptide to insert into membranes. Here we investigate how the negative charge of the membrane influences how pHLIP enters and exits the lipid bilayer. We found that electrostatic repulsion affected differently the membrane entry and exit of pHLIP for negatively charged membranes. As a consequence, a large hysteresis was observed. We propose this is not a consequence of structural changes but results from local changes in the environment of aspartic acids, shifting their pK values.

A previously reported multi-scale model for (ultra-)small-angle X-ray (USAXS/SAXS) and (very) small-angle neutron scattering (VSANS/SANS) of live Escherichia coli was revised on the basis of compositional/metabolomic and ultrastructural constraints. The cellular body is modeled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was replaced by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale. Specifically, this approach provides detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. The model was also successfully applied to E. coli K12, used for the authors' original modeling, as well as for two other E. coli strains. Significant differences were detected between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of the approach outlined here to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labeling agents.

Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH-dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane α-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes in vitro, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with timeresolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) displayed slow spontaneous flip-flop at 37 • C (half-time, t 1/2 = 140 h). However, inclusion of peptides, namely, gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide), accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to preformed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 h. Gramicidin, on the other hand, was preincorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g., adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.