Differential mechanisms for calcium-dependent protein/membrane association as evidenced from SPR-binding studies on supported biomimetic membranes
Résumé
In this work, two different types of supported biomimetic membranes were designed to study the membrane binding properties of two different proteins that both interact with cellular membranes in a calcium-dependent manner. The first one, neurocalcin, is a member of a subfamilly of EF-hand calcium-binding proteins that exhibit a calcium-myristoyl switch. The second protein is a bacterial toxin, the adenylate cyclase produced by Bordetella pertussis, the causative agent of whooping cough. The biomimetic membranes constructed in this study were either hybrid bilayer membranes or polymer-tethered membranes. Hemimembrane formation was obtained in two steps: a monolayer of 1-octadecanethiol or octadecyl-trichlorosilane was self-assembled on top of the gold or glass surface, respectively, and then the egg-phosphatidyl choline (PC) vesicle fused on the hydrophobic alkyl layer. Polymer-tethered membranes on solid support were obtained using N-hydroxysuccinimide (NHS)-terminated-poly(ethyleneglycol) (PEG)-phospholipids as anchoring molecules. Egg-PC/1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-poly(ethyleneglycol)-N-hydroxy-succinimide (DSPE-PEG-NHS) mixture liposomes were injected on the top of an amine grafted surface (cysteamine-coated gold or silanized glass); vesicles were linked to the surface and disrupted, leading to the formation of a bilayer. The biomimetic membrane constructions were followed by surface plasmon spectroscopy, while membrane fluidity and continuity were observed by fluorescence microscopy. Protein/membrane binding properties were determined by resonance surface plasmon measurements. The tethered bilayer, designed here, is very versatile as it can be adapted easily to different types of support. The results demonstrate the potentialities of such polymer-tethered artificial membranes for the study of proteins that insert into biological membranes such as toxins and/or integral membrane proteins.