Differential Mechanisms for Calcium-Dependent Protein/Membrane Association as Evidenced from SPR-Binding Studies on Supported Biomimetic Membranes

Abstract

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 octadecyltrichlorosilane 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.

Surface plasmon resonance in protein–membrane interactions

“…This was indeed observed in the case of coagulation factor VIII (Saenko et al, 2001). On the other hand, if the protein inserts deeper in the membrane and needs a transmembrane compartment, then no binding is observed in HBM, as shown by Rossi et al (2003) for adenylate cyclase from Bordetella pertussis which is able to translocate across the membranes in the presence of calcium.…”

Exploring the Membrane Mechanism of the Bioactive Peptaibol Ampullosporin A Using Lipid Monolayers and Supported Biomimetic Membranes

“…The measurements were performed in the configuration described in the previous studies [47, 48]. Optical thicknesses were determined according to Fresnel equations using the Winspall program (Max-Planck Institute for Polymer Research, Mainz, Germany).…”

“…A buffer rinsing step was performed in between each injection. The lipid diffusion coefficients were determined from FRAP experiments according to calculations previously published [47, 48]. …”

Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of Respiratory Infections Due toBordetella pertussisand OtherBordetellaSubspecies

“…The 1,300-aminoacid C-terminal domain mediates delivery of the catalytic domain into the cytoplasm of eukaryotic cells and possesses low but detectable hemolytic activity for sheep red blood cells (46,349,668). Amino acid sequence similarity between the Cterminal domain of CyaA, the hemolysins of E. coli (HlyA) and Actinobacillus pleuropneumoniae (HppA), and the leukotoxins of Pasteurella hemolytica (LktA) and Actinobacillus actinomycetemcomitans (AaLtA) places CyaA within a family of calcium-dependent, pore-forming cytotoxins known as RTX (repeats-in-toxin) toxins (659,672,676,813). Each of these toxins contains a tandem array of a nine amino acid repeat [LXGG XG(N/D)DX] that is thought to be involved in calcium binding (813).…”