Browsing by Author "Ross, Michaela"

By means of the quartz crystal microbalance (QCM) technique, the interaction of annexin Al with lipid membranes was quantified using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of a first octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer obtained from vesicle fusion. This experimental setup enabled us to determine for the first time rate constants and affinity constants of annexin Al binding to phosphatidylserine-containing layers as a function of the calcium ion concentration in solution and the cholesterol content within the outer leaflet of the solid-supported bilayer. The results reveal that a decrease in Ca2+ concentration from 1 mM to 100 muM significantly increases the rate of annexin A] binding to the membrane independent of the cholesterol content. However, the presence of cholesterol in the membrane altered the affinity constants considerably. While the association constant decreases with decreasing Ca2+ concentration in the case of 1-paimitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) membranes lacking cholesterol, it remains high in the presence of cholesterol.

By means of the quartz crystal microbalance (QCM) technique, we investigated the interaction of porcine heterotetrametric annexin A2t with solid supported lipid membranes. Dissociation and rate constants of annexin A2t binding to various lipid mixtures were determined as a function of Call concentrations in solution. In contrast to what has been observed for annexin A1, the binding affinity and kinetics of annexin A2t binding are not influenced by cholesterol. In the experimental setup chosen, the annexin A2t binding is strictly Ca2+-dependent and only affected by the amount of phosphatidylserine (PS) in the membrane and the Ca2+ concentration in solution. By Ca2+-titration experiments at constant annexin A2t concentration, we investigated the reversibility of annexin A2t adsorption and desorption. Surprisingly, Ca2+-titration curves display a significant hysteresis. Protein desorption curves starting from annexin A2t bound to the membrane at 1 mM CaCl2 exhibit high cooperativity with half-maximum Ca2+ concentrations in the submicromolar range. However, protein adsorption curves starting from an EGTA-containing solution with soluble annexin A2t always show two inflection points upon addition of Ca2+ ions. These two inflection points may be indicative of two protein populations differently bound to the solid-supported membrane. The ratio of these two annexin A2t populations depends on the amount of PS molecules and cholesterol in the membrane as well as on the Call concentration. We propose a model discussing the results obtained in terms of two binding sites differing in their affinity due to lipid rearrangement.

The first step in membrane aggregation mediated by the annexin A2-S100A10 heterotetrameric complex (annexin A2t) has been unraveled by in situ scanning force microscopy. It has been shown unambiguously that annexin A2t binds to solid-supported lipid bilayers simultaneously with both annexin A2 subunits.

A monomolecular protein adsorption was observed for the binding of annexin I to phosphatidylserine-enriched lipid domains. The interactions of this membrane-associated protein (red in the picture; green = N terminus, . = Ca(2+)-binding site) with phospholipid bilayers immobilized on mica were visualized by scanning force microscopy. Domain formation in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/-phosphoserine layers was induced by Ca(2+) ions rather than by the protein itself, as demonstrated by time-of-flight secondary-ion mass spectrometric analysis and lateral force microscopy.