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Journal for Biophysical Chemistry

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Addressable adsorption of lipid vesicles and subsequent protein interaction studies

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We demonstrate a convenient chip platform for the addressable immobilization of protein-loaded vesicles on a microarray for parallelized, high-throughput analysis of lipid-protein systems. Self-sorting of the vesicles on the microarray was achieved through DNA bar coding of the vesicles and their hybridization to complementary strands, which are preimmobilized in defined array positions on the chip. Imaging surface plasmon resonance in ellipsometric mode was used to monitor vesicle immobilization, protein tethering, protein-protein interactions, and chip regeneration. The immobilization strategy proved highly specific and stable and presents a mild method for the anchoring of vesicles to predefined areas of a surface, while unspecific adsorption to both noncomplementary regions and background areas is nonexistent or, alternatively, undetectable. Furthermore, histidine-tagged receptors have been stably and functionally immobilized via bis-nitrilotriacetic acid chelators already present in the vesicle membranes. It was discovered though that online loading of proteins to immobilized vesicles leads to cross contamination of previously loaded vesicles and that it was necessary to load the vesicles offline in order to obtain pure protein populations on the vesicles. We have used this cross-binding effect to our benefit by coimmobilizing two receptor subunits in different ratios on the vesicle surface and successfully demonstrated ternary complex formation with their ligand. This approach is suitable for mechanistic studies of complex multicomponent analyses involving membrane-bound systems.


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    The reference patches BSA and dextran show nonspecific adsorption of histidine-tagged receptors. This adsorption seems to be of a highly unstable nature as it bleeds completely off the surface with time. A related observation from the experiment is that when the vesicles were loaded with a relatively high concentration of receptor proteins, a certain loading level was passed, after which an unstable immobilization can be seen dissociation. However, the dissociation levels off at a value at which it is stable. When lower amounts of proteins were injected, below that value, the immobilization was observed to be stable from the start data not shown. We believe this to be the result of an interaction between the histidine-tagged and loosely associated ions (Ni2+) on the surface (i.e., the BSA, dextran, and c-DNA tags of the vesicles.

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    The B′ and D′ systems show a higher degree of repeatability in the signals compared to the A′ and C′ systems, which display a higher internal variance. This variation is an effect of the dispensing protocol; as the dispensing was manual and the location of the dispensing spots had to be revealed, water was evaporated on the array. Ideally, dispensing should occur as soon as the pattern is visible, but, in many cases, it got delayed and the formation of larger condensation droplets of water occurred before the spot was covered with the dispensing NA-ssDNA solution. Diffusion of proteins into the water droplets is relatively slow, which means that these areas of the spots will only partially be modified by NA-ssDNA and cannot fully interact with the vesicles. However, the variation remains consistent proportional throughout the whole time and is easily resolved by scaling as is shown ahead.

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    {au{gnG. L.} {fnZubay}}, {btBiochemistry}, {en4th} ed. {ei{fnBrown}}, {ei{fnDubuque}, {gnIA}}, ({dy1998}).

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Correspondence to Bo Liedberg.

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