Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter.

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 10(5) FomA proteins could be incorporated in a bilayer array with a total membrane area of 2mm(2) within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

Free-standing biomimetic polymer membrane imaged with atomic force microscopy.

Fluid polymeric biomimetic membranes are probed with atomic force microscopy (AFM) using probes with both normal tetrahedrally shaped tips and nanoneedle-shaped Ag(2)Ga rods. When using nanoneedle probes, the collected force volume data show three distinct membrane regions which match the expected membrane structure when spanning an aperture in a hydrophobic scaffold. The method used provides a general method for mapping attractive fluid surfaces. In particular, the nanoneedle probing allows for characterization of free-standing biomimetic membranes with thickness on the nanometer scale suspended over 300-µm-wide apertures, where the membranes are stable toward hundreds of nanoindentations without breakage.

Biomimetic triblock copolymer membrane arrays: a stable template for functional membrane proteins.

It is demonstrated that biomimetic stable triblock copolymer membrane arrays can be prepared using a scaffold containing 64 apertures of 300 microm diameter each. The membranes were made from a stock solution of block copolymers with decane as a solvent using a new deposition method. By using decane, we avoid low molecular weight solvents such as chloroform and toluene, which are strong protein denaturants. The membranes show a low ionic conductance and a long lifetime at room temperature. Contrast phase microscopy shows the presence of a polymer region delimited by a Plateau-Gibbs border similar to what is observed in black lipid membranes. The ion-channel gramicidin A was successfully incorporated into the membrane in a functional form.

Competitive protein adsorption to polymer surfaces from human serum.

Surface modification by "soft" plasma polymerisation to obtain a hydrophilic and non-fouling polymer surface has been validated using radioactive labelling. Adsorption to unmodified and modified polymer surfaces, from both single protein and human serum solutions, has been investigated. By using different radioisotopes, albumin and Immunoglobulin G (IgG) adsorption has been monitored simultaneously during competitive adsorption processes, which to our knowledge has not been reported in the literature before. Results show that albumin and IgG adsorption is dependent on adsorption time and on the presence and concentration of other proteins in bulk solutions during adsorption. Generally, lower albumin and IgG adsorption was observed on the modified and more hydrophilic polymer surfaces, but otherwise the modified and unmodified polymer surfaces showed the same adsorption characteristics.

Modeling the two-hybrid detector: experimental bias on protein interaction networks.

This work was done to investigate the two-hybrid experiment for finding protein-protein interactions to explain the asymmetry found in the experimental data, and to help screen the data for high confidence interactions. By looking at the bait-prey experimental setup the resulting protein interaction network can be examined as a directed network (bait --> prey). We have investigated two possible scenarios for the asymmetry in the directed network by developing a biochemical model for the protein-DNA and protein-protein bindings inside the living yeast. One scenario assumes a background activity of bait proteins acting even without the prey, the other scenario explores the asymmetry in the chemistry associated with the bait being automatically located in the right position on the DNA. We conclude that the latter model gives the best description of the observed asymmetry.

Protein aggregation and degradation during iodine labeling and its consequences for protein adsorption to biomaterials.

Protein adsorption on modified and unmodified polymer surfaces investigated through radiolabeling experiments showed a tendency for higher than expected albumin and immunoglobulin G (IgG) adsorption. Possible enhanced protein aggregation and degradation caused by the iodine labeling method used were analyzed through chromatography and spectroscopy techniques. Results show that the iodine labeling method using chloramine-T (CAT) as an oxidizing agent can cause both enhanced aggregation and fragmentation of proteins. Albumin shows an enhanced tendency to aggregate after iodine labeling using the CAT method, and higher amounts of fragmentation are observed for CAT-labeled IgG molecules relative to unlabeled IgG molecules as well as to IgG molecules labeled using the Iodo-Gen method. These results show that the widely applied method of radioisotope labeling for quantitative assessment of protein adsorption should be used with caution and preferably should be validated by a label-free methodology for each combination of radiolabel and protein. The results obtained in this study can be used to optimize investigation of protein adsorption on surfaces of materials for biomedical devices.