title: Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.
abstract: Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of membrane-bound macromolecules.
authors.affiliation: Department of Mechanical Engineering and Material Science, Duke University, Durham, North Carolina 27708-0300 USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Noppl-Simson
authors.forename: D
authors.initials: D
authors.lastname: Needham
mesh_terms: D000327:Adsorption; D001360:Avidin / Q000737:chemistry*; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D001710:Biotin / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004058:Diffusion; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D013499:Surface Properties
chemical_list: D003432:Cross-Linking Reagents; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008563:Membrane Lipids; D008565:Membrane Proteins; D001360:Avidin; D011092:Polyethylene Glycols; D001710:Biotin