Scrutiny of Annexin Al Mediated Membrane-Membrane Interaction by Means of a Thickness Shear Mode Resonator and Computer Simulations*

摘要

The dissipational quartz crystal microbalance (D-QCM) technology was applied to monitor the adsorption of vesicles to membrane-bound annexin Al by simultaneously reading out the shifts in resonance frequency and dissipation.Solid-supported membranes (SSMs) composed of a chemisorbed octanethiol monolayer and a physisorbed l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine monolayer were immobilized on the gold electrode of a 5 MHz quartz plate.Adsorption and desorption of annexin Al to the SSM was followed by means of the QCM technique.After nonbound annexin Al was removed from solution,the second membrane binding was monitored by the D-QCM technique,which allowed distinguishing between adsorbed and ruptured vesicles.The results show that vesicles stay always intact independent of the amount of bound annexin and the vesicle and buffer composition.It was shown that the vesicle adsorption process to membrane-bound annexin Al is fully irreversible and is mediated by two-dimensional annexin clusters.For N-terminally truncated annexin Al,a decrease in the amount of bound vesicles was observed,which might be the result of fewer binding sites presented by the annexin Al core.Supported by computer simulations,the results demonstrate that the vesicle adsorption process is electrostatically driven,but compared to those of sole electrostatic binding,the rate constants of adsorption are 1-2 orders of magnitude smaller,indicating the presence of a potential barrier.