Avtor/Urednik		Anderluh, Gregor; Razpotnik, Andrej; Podlesek, Zdravko; Maček, Peter; Separovic, Frances; Norton, Raymond S
Naslov		Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies
Tip		članek
Vir		J Mol Biol
Vol. in št.		Letnik 347
Leto izdaje		2005
Obseg		str. 27-39
Jezik		eng
Abstrakt		Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by sitedirected mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. T'he lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.
Deskriptorji		CNIDARIAN VENOMS PORINS CELL MEMBRANE PERMEABILITY MEMBRANES, ARTIFICIAL NUCLEAR MAGNETIC RESONANCE SEA ANEMONES ESCHERICHIA COLI MUTAGENESIS, SITE-DIRECTED SPHINGOMYELINS PHOSPHOLIPIDS PHOSPHATIDYLCHOLINES