Author/Editor		Malovrh, Petra; Barlič, Ariana; Podlesek, Zdravko; Maček, Peter; Menestrina, Gianfranco; Anderluh, Gregor
Title		Structure-function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein
Type		članek
Source		Biochem J
Vol. and No.		Letnik 346
Publication year		2000
Volume		str. 223-32
Language		eng
Abstract		Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three diflerent regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp45, EqtII Trp116/117 and EqtII Trp149, were prepared in an Escherichia coli expression system (here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein). They all possess a single intrinsic fluoresant centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp116/117 resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp45 and EqtII Trp149, was altered to a certain extent. EqtII Trp149 was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with 2H2O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp45 and EqtII Trp149 were more exposed to water, and also remained as such in the membrane-bound form.
Descriptors		CNIDARIAN VENOMS TRYPTOPHAN STRUCTURE-ACTIVITY RELATIONSHIP MEMBRANE LIPIDS SEA ANEMONES MUTATION ESCHERICHIA COLI PORINS ERYTHROCYTES MEMBRANES, ARTIFICIAL SPECTROSCOPY, FOURIER TRANSFORM INFRARED HEMOLYSIS