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 Lösche1,2 1Carnegie Mellon University, Pittsburgh, PA; 2NIST Center for Neutron Research, Gaithersburg, MD Motivation Biomembranes are complex structures com- posed of a large variety of lipids and proteins. These play important roles in cellular structure and function. Membrane Biophysics aims at investigating such functions using simplified model systems. We developed a robust membrane model – the tethered lipid bilayer membrane (tBLM) – which may incorporate lipid mixtures of controlled complexity and may incorporate asymmetric bilayers. Bilayers of mixed composition are required for a variety of protein-membrane inter- action studies. Here, we present the characteri- zation of these asymmetric bilayers using neutron reflection. Sample preparation / characterization Mixed tethered bilayer lipid membranes (tBLMs) are formed on gold-coated Si/SiOx wafers. The wafer is pre-incubated with a mixture of tether molecules (WC14, synthesized at NIST) and β-mercaptoethanol (βME), which forms a self-assembled monolayer (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 sub-membrane space. After SAM formation, the bilayer is incubated with a 10 mg/ml ethanolic lipid solution (which may be of mixed composition). “Rapid solvent exchange” with buffer is then used to complete the bilayer, forming the finished tBLM. In our hands, this preparation produces high-quality, insulating bilayer, as assessed by electrochemical impedance spectroscopy (EIS) and neutron reflection (NR). Fig. 1: Top: Molecular architecture of the WC14- based tBLM system (left) and a typical nSLD profile (right) of the fully hydrogenated lipid bilayer. Left: Schematic drawing of the advanced neutron diffractometer/reflectometer (AND/R) at the NCNR. The instrument is optimized for the study of biomembranes and biomembrane components Results Asymmetric tBLMs In order to quantify bilayer asymmetry by neutron reflec-tion, we used lipid mixtures with deuterated and hydrogen-ated chains. For a particular mixture of lipids, we observed that the nSLD of the outer lipid leaflet decreases and that of the inner lipid leaflet increases with time (i.e., it varies between consecutive measure- ments of the same tBLM at different solved contrasts). However, the average nSLD of the lipid leaflets remained constant. The results of one such experiment (70% hydrogenated DMPC, 30% deuter-ated DMPS and 3% cholesterol) is shown in Fig. 2. Fig. 2: Variation over time of the nSLDs of the leaflets in a tBLM (DMPC:d54- DMPS:cholesterol = 70:30:3) prepared on WC14:βME (3:7). Fig. 3: Average nSLD values (inner and outer leaflets) for tBLMs with different ratios of DMPC, DMPS and cholesterol. DMPC DMPS w/ cholesterol w/o cholesterol Fig. 4: Flipping rate dependence on the defect density of the bilayer. Lipid flip-flop The observation reported in Fig. 2 might be interpreted as an exchange of deuterated lipids (initially accumulated in the outer leaflet) and hydrogenated lipid (initially accumulated in the inner leaflet, with no net change in material densities of the two leaflets. However, we observe similar adjustments between the nSLDs of the two leaflets in tBLMs where there are only lipids with deuterated chains. While do not fully understand the structural basis of these rearrange- ments, we may evaluate their kinetics by tracking the rates for molecular transfer between the leaflets. We define the transfer rate as the number of lipids flipping from the outer to the inner leaflet per hour and per unit area (assumed to be ~ 60 Å2). This is equivalent to the probability of any given lipid flipping within one hour. Fig. 4 shows that low defect densities (determined as residual water in the plane of the bilayer) correlate with low flipping rates. At higher defect densities, we observe increased flipping rates in DMPC/DMPS tBLMs without cholesterol, but not in tBLMs with cholesterol. Other lipid mixtures have also been investigated. Each of them show a similar trend of deuterated lipids going from outer lipid leaflet to inner lipid leaflet (see Table 1). Lipids investigated deuteration fraction defect density d54-DMPG:chol (100:1) 1 .1403 d54-DMPC:DMPS (50:50) .5 .1136 d54-DMPC:DMPS (70:30) .7 .2032 d54-DMPC:DMPS (85:15) .85 .2759 DMPC:d54-DMPS:chol (70:30:3) .3 .0151 d54-DMPC:DMPS:chol (70:30:3) .0049 d54-DMPC .0231 d54-DMPC:chol (100:1) .0622 d54-DMPC:chol (100:3) d31-POPC .0042 Table 1: A list of the lipids investigated for asymmetry. These are well prepared tBLMs with low defect density. Conclusions tBLMs of different lipid compositions may be prepared in a con- trolled way. The composition of the lipid leaflets can be precisely determined by neutron reflection using isotopic contrast. Bilayers formed from lipid mixtures within the tBLM systems are generally asymmetric, and show lipid flip-flop. The lipid flipping rate depends on defect density in the system. Cholesterol incorporates into the bilayers and stabilizes them by slowing lipid flip-flop. Acknowledgments Support by the National Institute of Standards and Technology in providing the neutron research facilities used in this work is gratefully acknowledged. This research was supported by the American Health Assistance Foundation (grant # A2008-307), the NIH (grant# 1P01 AG032131, and the Department of Commerce (MSE grant #70NANB8H8009). 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. Supramolecular Structures Lab Center for Neutron Research