Mixed self-assembled monolayers (SAMs) of β-mercaptoethanol and the new synthetic lipid 1,2-di-O-palmityl-3-[ω-mercapto-nona(ethylene oxide) glycerol], FC16, were investigated for their ability to form sparsely-tethered bilayer lipid membranes (stBLMs) completed with various phospholipids. We investigated the structural and functional properties of FC16-based stBLMs and compared these to stBLMs prepared using a previously characterized synthetic lipid, 1,2-di-O-myristyl-3-[ω-mercaptohexa(ethylene oxide) glycerol] (WC14). FC16-based stBLMs show increased resistivity to ion transfer and an increase in the submembrane space of ≈ 0.5 nm. Importantly, FC16-based stBLMs formed well-defined, complete bilayers with charged phospholipids such as POPG. In these, POPG, incorporates into the outer monolayer leaflet in the same ratio as in the immersion solution, but is excluded from the inner leaflet. In all cases we investigated thus far, the area densities of the lipids within the bilayers were on average close to those in free bilayer membranes. For charged phospholipids, FC16 appears to provide a distinct advantage over WC14 for the formation of well-defined stBLMs.-2 -

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P2 were determined to be Kd∼12 µM and 0.4 µM, respectively, and Kd∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P2 and an increased apparent affinity to PI(3,4,5)P3, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P2 and no synergy in its binding with PS and PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.

The self-assembled monolayers (SAMs) of the new lipidic anchor molecule HC18 [Z 20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol], and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection adsorption infrared spectroscopy (RAIRS), electrochemical impedance spectroscopy (EIS), and evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients, relative to previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange (RSE) or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore forming toxin, α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicates HC18 results in tBLMs with improved physical properties for incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution is imperative.