Abstract
Planar lipid bilayers on sensor chip surfaces have become useful tools to study membrane bound processes by surface plasmon resonance spectroscopy. We immobilized phospholipids on sensor chips by different approaches. First, a self-assembled monolayer of octadecylmercaptan was formed on a blank gold surface and subsequent addition of phospholipids led to formation of a heterobilayer. Second, a self-assembled monolayer of mercaptoundecanoic acid was formed on a gold surface, the carboxy groups of mercaptoundecanoic acid were activated and covalently linked to phosphatidylethanolamine. Addition of phospholipids then led to a bilayer with phosphatidylethanolamine as the lower leaflet. Third, a hydrophobic sensor chip (L1, BIAcore) was used as a binding matrix for phospholipids. These lipid surfaces were tested, whether they are suitable to study proteinamembrane interactions. As biological test system we used the Ca2+-myristoyl-switch of the neuronal Ca2+-binding protein recoverin. All three surfaces were sufficiently stable to monitor the Ca2+-dependent binding of recoverin to membranes.
Highlights
Evanescent wave biosensors have attained wide interest in the last years
Bilayers were used to study the Ca2+-dependent membrane association of recoverin [6,15], a myristoylated Ca2+-binding protein, that undergoes a Ca2+-induced structural change with the exposition of its covalently attached myristoyl group [2,7,23]. Since this process represents a reversible binding event to membranes and since it is controlled by a change in free Ca2+-concentration, it is ideally suited to test the applicability of different lipid layers on solid supports
First a hydrophobic self assembled monolayer (SAM) consisting of octadecylmercaptan was formed on the blank gold surface and lipids or lipid mixtures were flushed over the surface in surface plasmon resonance (SPR) running buffer
Summary
Evanescent wave biosensors have attained wide interest in the last years. They employ surface plasmon resonance (SPR) spectroscopy to study the interaction of biological macromolecules, in particular the association and dissociation of proteins and peptides [11,20,21]. Bilayers were used to study the Ca2+-dependent membrane association of recoverin [6,15], a myristoylated Ca2+-binding protein, that undergoes a Ca2+-induced structural change with the exposition of its covalently attached myristoyl group [2,7,23]. Since this process represents a reversible binding event to membranes and since it is controlled by a change in free Ca2+-concentration, it is ideally suited to test the applicability of different lipid layers on solid supports
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