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.
Ross
Michaela
M
Institut für Biochemie, Westfälische Wilhelms-Universität, Wilhelm-Klemm-Strasse 2, 48149 Münster, Germany.
Gerke
Volker
V
Steinem
Claudia
C
D000327:Adsorption; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D003460:Crystallization; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D009991:Oscillometry / Q000295:instrumentation / Q000379:methods; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011791:Quartz; D013552:Swine
D017306:Annexin A2; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010743:Phospholipids; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D011791:Quartz; D002784:Cholesterol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine