application of different lipid surfaces to monitor protein–membrane interactions by surface plasmon resonance spectroscopy应用不同的脂质表面通过表面等离子体共振光谱监测蛋白质膜的相互作用.pdfVIP

Spectroscopy 16 (2002) 271–279 271 IOS Press Application of different lipid surfaces to monitor protein–membrane interactions by surface plasmon resonance spectroscopy Torsten Fischer, a,1 Ivan I. Senin b , Pavel P. Philippov b and Karl-Wilhelm Koch a,∗ a Institut für Biologische Informationsverarbeitung 1, Forschungszentrum Jülich, D-52425 Jülich, Germany b A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia 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 protein–membrane 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. Abbreviations ROS, bovine rod outer segments; PMSF, phenylmethyl-sulfonylfluoride; SPR, surface plasmon res- onance spectroscopy; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; SAM, self-assembled monolayer; NHS, N-hydroxysuccinimid; EDC, N-ethyl-N -[(