silicon and 1-palmitoyl-2-oleoylglycero-3-phosphoserine

Interactions of soluble proteins with the cell membrane are critical within the blood coagulation cascade. Of particular interest are the interactions of γ-carboxyglutamic acid-rich domain-containing clotting proteins with lipids. Variability among conventional analytical methods presents challenges for comparing clotting protein-lipid interactions. Most previous studies have investigated only a single clotting protein and lipid composition and have yielded widely different binding constants. Herein, we demonstrate that a combination of lipid bilayer nanodiscs and a multiplexed silicon photonic analysis technology enables high-throughput probing of many protein-lipid interactions among blood-clotting proteins. This approach allowed direct comparison of the binding constants of prothrombin, factor X, activated factor VII, and activated protein C to seven different binary lipid compositions. In a single experiment, the binding constants of one protein interacting with all lipid compositions were simultaneously determined. A simple surface regeneration then facilitated similar binding measurements for three other coagulation proteins. As expected, our results indicated that all proteins exhibit tighter binding (lower

Topics: Factor VIIa; Factor X; High-Throughput Screening Assays; Humans; Kinetics; Lipid Bilayers; Nanostructures; Optical Phenomena; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylserines; Protein Array Analysis; Protein C; Prothrombin; Recombinant Proteins; Silicon

The assembly of electrically addressable, planar supported bilayers composed of biologically relevant lipids, such as those used in vesicular systems, will greatly enhance the experimental capabilities in membrane and membrane protein research. Here we assess the electrical properties of bilayers composed of a wide range of physiologically relevant lipids and lipid combinations. We demonstrate that robust, biologically relevant, planar supported bilayers with high resistance composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 25 mol % cholesterol can be constructed with high reproducibility. Furthermore, to enable studies of pore-forming peptides, which are commonly cationic, we demonstrate the construction of bilayers with biologically relevant outer leaflets incorporating up to 10 mol % negatively charged lipids. Unique features of the platform are that (1) the substrate is commercially available, atomically smooth, single-crystal silicon, (2) the polymer cushion allows for the natural incorporation of membrane proteins, and (3) the platform is highly reproducible.

Topics: Cholesterol; Electricity; Lipid Bilayers; Membrane Proteins; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Reproducibility of Results; Silicon; Surface Properties

Specular neutron reflectivity was used to study the time course and nature of the interaction of the positively charged, peripheral membrane protein cytochrome c with supported bilayers of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) containing the anionic lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylserine (POPS). The supported bilayers were prepared by deposition on silicon blocks of two monolayers of DOPC, the second of which contained either 10 or 20 mol % POPS at surface pressures of either 15 or 20 mN/m using a combination of Langmuir-Blodgett and Schaefer deposition techniques. Each supported bilayer was initially characterized by specular neutron reflectivity using subphases of 10 mM NaCl aqueous solutions. Regardless of POPS content and bilayer deposition pressure, the molecular architecture of the bilayers was similar. The addition of cytochrome c resulted in an almost immediate change in reflectivity, which was well modeled by assuming that an additional layer was present next to the outer leaflet of the bilayer. The thickness of this layer, which contained the volume fraction of approximately 15% protein, was approximately 30 A (consistent with the cross-section of a single cytochrome c molecule). The addition of cytochrome c to the subphase also resulted in a change in the structure of the phospholipid bilayer, suggesting some penetration of cytochrome c into the bilayer. Specular neutron reflectivity studies after careful washing with solvent showed that although most of the protein was washed off by flushing 10 mM NaCl D2O through the cell a small amount remained both within the bilayer and bound to the membrane surface.

Topics: Cell Membrane; Cytochromes c; Lipid Bilayers; Neutron Diffraction; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Silicon; Sodium Chloride