1. Surface-supported Bilayer Lipid Membranes from Lipid Mixtures: Composition and Transverse Molecular Flip-Flop Prabhanshu Shekhar 1, Frank Heinrich 1,2, Hirsh Nanda 2, Mathias Lösche 1,2 1 Carnegie Mellon University, Pittsburgh, PA; 2 NIST Center for Neutron Research, Gaithersburg, MD Motivation Biomembranes are complex structures composed of a large variety of lipids and proteins. These play an important role in cellular structure and function. Lipid bilayers of mixed composition are required for a variety of studies of the protein-membrane interactions. For example, the GAG proteins, associated with HIV virus assembly in infected cells, interact with anionic lipids in membranes. We have developed a robust model membrane system which has been used to form asymmetric bilayers. Here, we present the characterization of these asymmetric bilayers using neutron reflectivity. DMPC DMPS Asymmetric tBLMs In order to see the asymmetry in the bilayer using neutron reflectivity, we used deuterated lipids mixed with hydrogenated lipids. For a particular mixture of lipids, we found that the nSLD of the outer lipid leaflet goes down and that of inner lipid leaflet goes up with time. The average nSLD value of the lipid leaflets remains constant. The results of one such experiment (70% hydrogenated DMPC, 30% deuterated DMPS and 3% cholesterol) is shown in Fig 2. Similar results were obtained for different compositions of DMPC and DMPS mixtures. Conclusions We can prepare bilayers of different compositions. The composition of the lipid leaflets can be determined precisely. The bilayesr formed from lipid mixtures are generally asymmetric and show flip-flop. Flipping rate is dependent on the defect density of the system. Cholesterol incorporates into the bilayer and stabilizes it, thereby slowing the flip-flop process. Tethered mixed bilayer lipid membranes (tBLMs) are formed on gold-coated Si/SiOx wafers. The wafer is pre- incubated with a mixture of tether molecule (WC14, synthesized at NIST) and β-mercaptoethanol, which forms a monolayer on gold by self-assembly (SAM). WC14 has two alkyl tails linked through a glycerol to a thiolated hexa(ethylene oxide) “spacer”. The spacer separates the membrane from the gold surface, providing ≈ 20 Å thick, water-filled submembrane space. After SAM formation, the bilayer is incubated with a 10 mg/ml lipid/cholesterol solution in methanol or ethanol. The tBLM is completed by “rapid solvent exchange”. The rapid solvent exchange was demonstrated to form nearly defect-free bilayers. Alternative preparation techniques (vesicle fusion, Langmuir Blodgett transfer, etc) produce incomplete bilayers in most cases. Fig 1: Molecular architecture of the WC14 tBLM system and a typical nSLD profile of the fully hydrogenated lipid bilayer. Acknowledgments This research was supported by….. References D.J. McGillivray, et al., Molecular-scale structural and fuctional characterization of sparsely tethered bilayer lipid membranes. Biointerphases 2 (2007), 21-33 F. Heinrich, et al., A new lipid anchor for sparsely-tethered bilayer lipid membranes. Langmuir 25 (2009), 4219-4229 G. Valincius, et al., Soluble amyloid β oligomers affect dielectric membrane properties by bilayer insertion and domain formation: Implications for cell toxicity. Biophys. J. 95 (2008), 4845-4861. Fig 2: Variation of the nSLD of the leaflets with time for a DMPC:d 54 -DMPS:cholesterol (70:30:3) mixture on WC14:βME (3:7) system. Fig 3: Plot of average value of the nSLD of inner and outer lipid leaflets for various experiments done using different ratios of DMPC, DMPS and cholesterol. Sample Prep / Neutron Reflectivity Lipid Flip-Flop The observation in fig. 2 can be extended to find the rate at which the deuterated lipids move from outer lipid leaflet to the inner lipid leaflet. We define this rate as the number of lipids flipping from the outer into the inner lipid leaflet per hour and per 60Å 2. This is equivalent to the probability of any given lipid (area per lipid 60Å 2 ) flipping within one hour. The plot (Fig 4) shows that at low defect densities we observe low flipping rates. At higher defect densities we observe higher flipping rates in bilayers of DMPC/DMPS without cholesterol, but the flipping rate is considerably lower in bilayers with cholesterol. Other lipid mixtures have also been tried. Each of them show a similar trend of deuterated lipids going from outer lipid leaflet to inner lipid leaflet. Fig 4: Flipping rate dependence on the defect density of the bilayer. w/ cholesterol w/o cholesterol Results