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The compositional asymmetry of biological membranes has attracted significant attention over the last decade. Harboring more differences from symmetric membranes than previously appreciated, asymmetric bilayers have proven quite challenging to study with familiar concepts and techniques, leaving many unanswered questions about the reach of the asymmetry effects. One particular area of active research is the computational investigation of composition- and number-asymmetric lipid bilayers with molecular dynamics (MD) simulations. Offering a high level of detail into the organization and properties of the simulated systems, MD has emerged as an indispensable tool in the study of membrane asymmetry. However, the realization that results depend heavily on the protocol used for constructing the asymmetric bilayer models has sparked an ongoing debate about how to choose the most appropriate approach. Here we discuss the underlying source of the discrepant results and review the existing methods for creating asymmetric bilayers for MD simulations. Considering the available data, we argue that each method is well suited for specific applications and hence there is no single best approach. Instead, the choice of a construction protocol—and consequently, its perceived accuracy—must be based primarily on the scientific question that the simulations are designed to address.

 

We addressed the frequent occurrence of mixed-chain lipids in biological membranes and their impact on membrane structure by studying several chain-asymmetric phosphatidylcholines and the highly asymmetric milk sphingomyelin. Specifically, we report trans-membrane structures of the corresponding fluid lamellar phases using small-angle X-ray and neutron scattering, which were jointly analyzed in terms of a membrane composition-specific model, including a headgroup hydration shell. Focusing on terminal methyl groups at the bilayer center, we found a linear relation between hydrocarbon chain length mismatch and the methyl-overlap for phosphatidylcholines, and a non-negligible impact of the glycerol backbone-tilting, letting the sn1-chain penetrate deeper into the opposing leaflet by half a CH2 group. That is, penetration-depth differences due to the ester-linked hydrocarbons at the glycerol backbone, previously reported for gel phase structures, also extend to the more relevant physiological fluid phase, but are significantly reduced. Moreover, milk sphingomyelin was found to follow the same linear relationship suggesting a similar tilt of the sphingosine backbone. Complementarily performed molecular dynamics simulations revealed that there is always a part of the lipid tails bending back, even if there is a high interdigitation with the opposing chains. The extent of this back-bending was similar to that in chain symmetric bilayers. For both cases of adaptation to chain length mismatch, chain-asymmetry has a large impact on hydrocarbon chain ordering, inducing disorder in the longer of the two hydrocarbons. 

It is well known that the lipid distribution in the bilayer leaflets of mammalian plasma membranes (PMs) is not symmetric. Despite this, model membrane studies have largely relied on chemically symmetric model membranes for the study of lipid–lipid and lipid–protein interactions. This is primarily due to the difficulty in preparing stable, asymmetric model membranes that are amenable to biophysical studies. However, in the last 20 years, efforts have been made in producing more biologically faithful model membranes. Here, we review several recently developed experimental and computational techniques for the robust generation of asymmetric model membranes and highlight a new and particularly promising technique to study membrane asymmetry. 

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.

 

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.