Coupling of Transverse and Lateral Structure in Asymmetric Lipid Bilayers

All biological cells are bordered a plasma membrane, which enables diverse physiological functions, including cellular communication and selective material transport into or out of the cell. The molecular composition of these outer membranes is complex involving as a central element a lipid/protein bilayer. Lipids are known to form the structural matrix of this layer embedding proteins with specific functions (e.g. pumps, ion channels, receptors), but are increasingly recognized for their functional role. For example, lipid disorder has been implicated in diverse diseases including cancer, diabetes type II, or Parkinson to name but a few. One of their physiological roles is to assist the formation of lipid/protein platforms, known as membrane rafts, which fulfill specific functions. Consequently membrane lipids are not equally distributed laterally, but segregated into certain domains. Moreover, plasma membranes typically display also transbilayer asymmetry, i.e. lipids are not homogeneously distributed within the two membrane leaflets. Mammalian plasma membranes, for example, actively sequester nearly all of its sphingomyelinand phosphatidylcholine lipids within theouterleaflet,while phosphatidylethanolamineand the negativelychargedlipids phosphatidylserineand phosphatidylinositol are found in the inner leaflet. Biophysical studies on artificial membranes mimicking plasma membrane have yielded significant insight on the active role of membrane lipids. For example, model membranes composed of lipids exclusively found in the outer membrane leaflet show the formation of domains. Interestingly, when repeating the same experiments with lipids of the inner leaflet no domains are observed. However, when constructing asymmetric lipid membranes with domain-forming lipids in the outer and non- domain-forming lipids in the inner leaflet, segregation of inner membrane lipids was observed. Hence, there must be a coupling mechanism which may originate from hydrocarbon chain interdigitation, cholesterol flip-flop, or curvature-tension related mechanisms. Understanding this transmembrane coupling is a key to our understanding of membrane function. Experimental studies on artificial asymmetric bilayers are sparse and consequently no clear-cut picture of transversal and later coupling in asymmetric membranes is available at present. Most recent developments in sample preparation allow high-resolution structural studies via combination of x-ray and neutron scattering experiments with molecular dynamics simulations. Thereby we will specifically study the nature of transmembrane coupling, determine the structural properties of domains in asymmetric bilayers and measure the preferred location of cholesterol in both membrane leaflets. We have assembled an international consortium of key researchers that will team-up to meet the goals of the project. It is expected that the proposed work will enable several future studies related to the influence of membrane asymmetry on protein function and the role of membrane active compounds, such as medical drugs.

Asymmetry is a central feature of biological systems on the molecular to macromolecular level. In particular biological membranes, which are involved in numerous physiological processes (e.g. signaling, communication, material transport) are composed of diverse proteins and phospholipids which are for most membrane distributed asymmetrically across the bilayer. Moreover, cells spend a significant amount of energy to establish and maintain lipid asymmetry using adenosine triphosphate dependent proteins (flippases, floppases). Mammalian plasma membranes for example are composed of an outer leaflet enriched in charge neutral phosphatidylcholine and sphingomyelin, whereas phosphatidylethanolamine and anionic phosphatidylserine are confined to the inner leaflet. Exposure of phosphatidylserine to the outer leaflet may result in cancer, programmed cell death (apoptosis) or coagulation of blood platelets. Besides the importance of maintaining phosphatidylserine in the inner leaflet, little is known about the need to establish asymmetry for other lipid species. Closely related to this the question of the coupling of both membrane leaflets, which is important for transmembrane signaling events, e.g. to recruit certain cytosolic proteins to the inner cell surface. The latter aspect was considered closely in the present project. In particular, we designed simplified artificial mimics of membranes with asymmetrically distributed lipids by advancing preparation protocols to produce 100 nm large asymmetric vesicles amenable to diverse biophysical techniques, including small-angle neutron and X-ray scattering (SANS, SAXS), solution nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) or cryo transmission electron microscopy (cryo-TEM), which allowed their detailed characterization. We also developed the analysis technique for combined SAXS and SANS experiments. The joint analysis considers the different contrasts achievable by the two techniques, which allows to retrieve leaflet specific structural information, such as e.g. the lipid packing or the leaflet thickness, with high structural accuracy. Two types of systems were studied: asymmetric vesicles composed of (i) lipids with the same head group, but different hydrocarbon chain and (ii) lipids with identical hydrocarbon chain composition but differing lipid headgroups. For the first system, we observed a partial fluidization of gel-like domains in the outer leaflet due to interactions with a fluid inner leaflet. The second system turned out to be even more intricate. Inner and outer leaflets were only coupled when the lipid with the smaller headgroup was enriched in the inner leaflet, but not for the opposite system, i.e. when it was placed in the outer leaflet. The latter finding coincides with the location of these lipids in biological membranes and suggests a novel mechanism for bilayer coupling based on the lipids molecular shape.