Author/Editor		Barlič, Ariana
Title		Interakcije ekvinatoksina II iz morske vetrnice Actinia equina z lipidnimi membranami
Translated title		Interactions of equinatoxin II from sea anemone Actinia equina with lipid membranes
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2000
Volume		str. 72
Language		slo
Abstract		Equinatoxin II (EqtII) is a basic protein from a sea anemone (Actinia equina L.), composed of 179 amino acids. It belongs to a large group of cytolytic toxins, capable of forming pores in biological membranes and artificial lipid bilayers. Despite the fact that molecule is relatively well characterized, details of its insertion into membrane and pore-formation are not known yet. To get more insight into these interactions, EqtII's tryptophan mutants had been produced and used in studies with model membrane systems. For the first time, atomic force microscopy was employed to observe oligomeric assemblies and ring-shaped structures of EqtII on supported planar lipid bilayers. Intrinsic fluorescence measurements of native EqtII, which contains five Trp residues, and EqtII mutants Trp45, Trp116.117 and Trp149 (number indicates preserved tryptophan residue; all others were substituted by Phe) showed transfer of residues Trp116 and Trp117 into the lipid bilayer upon membrane binding. Since residue Trp112 is located in immediate vicinity, we propose transfer of the whole hydrophobic cluster (residues 112-117) into the membrane. By the means of fluorescence resonance energy transfer we estimated location of residues Trp116 and Trp117 within the membrane. They are located approximately 2,2 nm from the center of the hydrophobic core, in lipid/water interface, thus resembling other membrane active proteins. Further characterization of the mutant Trp116.117 included binding studies to SUVs, measured by fluorescence anisotropy. We found out that the mutant binds almost 10x stronger than the native toxin. Accordina to ANS fluorescence measurements. one reason for this could be in a more "relaxed" conformation of the molecule, although secondary structural elements are preserved. The mutant also showed a higher binding affinity for homogenous lipid surfaces. (Abstract truncated at 2000 characters)
Descriptors		SEA ANEMONES CNIDARIAN VENOMS MEMBRANES, ARTIFICIAL LIPID BILAYERS MEMBRANE LIPIDS MICROSCOPY, ATOMIC FORCE TRYPTOPHAN LIPOSOMES ESCHERICHIA COLI TRANSFORMATION, GENETIC SPECTROMETRY, FLUORESCENCE