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Claudia Steinem

Publications and source records attributed to Claudia Steinem.

4 recordsLinked to original sources
PubMed2003

Membrane composition affects the reversibility of annexin A2t binding to solid supported membranes: a QCM study.

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 Ca2+ 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 Ca2+ concentration. We propose a model discussing the results obtained in terms of two binding sites differing in their affinity due to lipid rearrangement.

Adsorption↗
PubMed2002

Kinetics and thermodynamics of annexin A1 binding to solid-supported membranes: a QCM study.

By means of the quartz crystal microbalance (QCM) technique, the interaction of annexin A1 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 A1 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 Ca(2+) concentration from 1 mM to 100 microM significantly increases the rate of annexin A1 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 Ca(2+) concentration in the case of 1-palmitoyl-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.

Annexins↗
PubMed2002

Quantification of the Raf-C1 interaction with solid-supported bilayers.

By use of the quartz crystal microbalance technique, the interaction of the Raf-Ras binding domain (RafRBD) and the cysteine-rich domain Raf-C1 with lipids was quantified by using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of an initial octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer varying in its lipid composition as the outermost layer. The integrity of bilayer preparation was monitored by impedance spectroscopy. For binding experiments, a protein construct comprising the RafRBD and Raf-C1 linked to the maltose binding protein and a His tag, termed MBP-Raf-C1, was used. Dissociation constants and rate constants of the association and dissociation were obtained for various 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS) lipid mixtures. Independently of the phosphatidylserine (PS) content, the dissociation constants were in the order of 5x10(-7) M, while the on-rate constants were in the range of 2x10(3) (M s)(-1) and the off-rate constants in the range of 1x10(-3) s(-1). The maximum frequency shift increased significantly with increasing amounts of DMPS; this indicates that this negatively charged lipid is the primary binding site for MBP-Raf-C1. Exchange of DMPS for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) did not alter the thermodynamics and kinetics of protein binding, which implies that the protein interaction is mainly electrostatically driven. Scanning force microscopy (SFM) was employed to render protein adsorption visible and to confirm the assumption of a protein monolayer on the lipid layer. SFM images clearly revealed that the protein binds preferentially, but not solely, to negatively charged phosphatidylserine headgroups. We hypothesize that PS-enriched domains are initial binding sites with high affinity for Raf-C1, but that lateral interactions may account for protein domain growth.

Adsorption↗