A New Lipid Anchor for Sparsely Tethered Bilayer Lipid Membranes

Abstract

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 -

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

“…Dashed red lines indicate 68% confidence intervals for the free-form protein CVO profiles. C, probability distribution, determined from Monte Carlo data resampling (25), of myrG55 orientations (,) with respect to the bilayer normal. Only the half-sphere 0 Յ Յ 90°is shown.…”

“…4C, inset) were chosen to coincide with the longest ( 0 ) and second longest ( 0 ) principal axes of the mass distribution in the crystal structure. Parameter space was efficiently searched with a Monte-Carlo Markov Chain algorithm, and provided quantitative measures of parameter and neutron scattering length density confidence limits, as well as parameter cross-correlations (25). The crystal structure used in the refinement was reported for a truncated protein, GRASP55 , and also lacks the N-myristoylation and the C-terminal decapeptide and His tag.…”

“…The aforementioned stBLMs feature a nanometer-thick hydrated layer between the solid surface and the lipid bilayer (19,25) that keeps the bilayer in a physiologically relevant inplane fluid state (29). We used purified preparations of the N-myristoylated and unmyristoylated GRASP domain of GRASP55 (residues 1-208).…”

“…Neutron scattering is nondestructive, which allows all measurements to be conducted on the same sample area under identical conditions (19,35). These measurements are then evaluated in reference to each other (24,25) to obtain a unique, highly resolved model of the protein-membrane complex. Two complementing approaches to data modeling were FIGURE 3.…”

Interrogating protonated/deuterated fibronectin fragment layers adsorbed to titania by neutron reflectivity and their concomitant control over cell adhesion

“…The final data fits were then analysed via a Monte Carlo resampling method [23,24]. In this procedure, 1000 new datasets, based upon the original with Gaussian noise based upon the counting statistic added, are individually fitted.…”