We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 AE 0.5 Å for the investigated lipid compositions.curvature energy | hydration force | lipid bilayer | E xocytosis, intracellular transport, neurotransmission, fertilization, or viral entry, require that two membranes merge into one. This event, membrane fusion, involves a complex interplay of different membrane lipids, proteins, and water molecules on length scales of few nm. Following the "lipidic pore hypothesis", it is now well accepted that membrane fusion involves a sequence of lipidic nonbilayer intermediates, whose formation is catalyzed and guided by a specialized protein machinery (1, 2). A stage termed "hemifusion" in which the lipids of the outer leaflets of two membrane-enclosed compartments about to fuse can mix, whereas the inner leaflets and the enclosed content remain unaltered (3), has been confirmed by a variety of methods including conical electron tomography (4) and, more indirectly; e.g., by electron spin resonance (5) and fluorescence recovery after photobleaching (6). Studying the fusion of protein-free bilayers of well defined lipid composition can contribute useful insights into the physical principles governing the merger of their more complex biological counterparts and clarify the effect of individual lipid species.The first connection between two lipid bilayers is the so-called stalk sketched in Fig. 1A (7). The proximal lipid monolayers have merged into one strongly curved monolayer, whereas the distal ones are still separated and intact. A persistent problem in membrane biophysics is to determine the precise structure of stalks and the free energy barrier for stalk formation. In a long series of papers (8-16), this determination has been attempted within the framework of the continuum theory of membrane elasticity (17, 18). More recently, with the advent of sufficient computational power, simulations including molecular details have become feasible [e.g. (19, 20) and references therein]. While stalk formation between lipid bilayers in close contact is generally accepted as the initial step in possibly all membrane fusion reactions (7), the subsequent stages from stalk to complete fusion are still debated and different pathways may exist (21)....

We have developed an X-ray scattering setup which allows to study membrane fusion intermediates or other nonlamellar lipid mesophases by laboratory-scale X-ray sources alone, thus taking advantage of unrestricted beamtime compared to synchrotron sources. We report results of a study of pure lipid bilayers and phospholipid/cholesterol binary mixtures. Stalks, putative intermediate structures occurring during the membrane fusion process, can clearly be identified from reconstructed electron density maps. Phase diagrams of the lyotropic phase behavior of DOPC/cholesterol and DPhPC/cholesterol samples are presented. If cholesterol is present in moderate concentrations, it can substantially promote the formation of stalks at higher degree of hydration. In addition, a possibly new phase in DOPC/cholesterol is found at high cholesterol content in the low humidity range.

We have studied the acyl-chain conformation in stalk phases of model membranes by x-ray diffraction from oriented samples. As an equilibrium lipid phase induced by dehydration, the stalk or rhombohedral phase exhibits lipidic passages (stalks) between adjacent bilayers, representing a presumed intermediate state in membrane fusion. From the detailed analysis of the acyl-chain correlation peak, we deduce the structural parameters of the acyl-chain fluid above, at, and below the transition from the lamellar to rhombohedral state, at the molecular level.

We have carried out time resolved stroboscopic diffraction experiments on standing surface acoustic waves (SAWs) of Rayleigh type on a LiNbO3 substrate. A novel timing system has been developed and commissioned at the storage ring Petra III of Desy, allowing for phase locked stroboscopic diffraction experiments applicable to a broad range of timescales and experimental conditions. The combination of atomic structural resolution with temporal resolution on the picosecond time scale allows for the observation of the atomistic displacements for each time (or phase) point within the SAW period. A seamless transition between dynamical and kinematic scattering regimes as a function of the instantaneous surface amplitude induced by the standing SAW is observed. The interpretation and control of the experiment, in particular disentangling the diffraction effects (kinematic to dynamical diffraction regime) from possible non-linear surface effects is unambiguously enabled by the precise control of phase between the standing SAW and the synchrotron bunches. The example illustrates the great flexibility and universality of the presented timing system, opening up new opportunities for a broad range of time resolved experiments

We have studied the packing and collective dynamics of the phospholipid acyl chains in a model membrane composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and cholesterol in varied phase state. After a structural characterization of this two-component model bilayer using X-ray reflectivity, we have carried out coherent inelastic neutron scattering to investigate the chain dynamics. Both DMPC/cholesterol membranes exhibited much sharper and more pronounced low-energy inelastic excitations than a pure DMPC membrane. In the high-energy regime above 10 meV, the insertion of cholesterol into the membrane was found to shift the position of the inelastic excitation towards values otherwise found in the pure lipids gel phase. Thus, the dissipative collective short-range dynamics of the acyl chains is strongly influenced by the presence of cholesterol.