Author/Editor		Malovrh, Petra
Title		Strukturno-funkcionalne lastnosti triptonskih in cisteinskih mutant ekvinatoksina II iz morske veternice Actinia equina
Translated title		Structure-functional characteristics of tryptophan and cysteine mutants of equinatoxin II from the sea anemone Actinia equina
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2001
Volume		str. 109
Language		slo
Abstract		EqtII, a 179 amino acid residue protein, isolated from the sea anemone Actinia equina L., is a member of eukaryotic pore-forming toxins. It belongs to a group of cytolysins, named actinoporins, which are found exclusively in sea anemones (Actiniaria). They have molecular masses ranging from 10-20 kDa and are basic, single-chain proteins, devoid of cysteine residues, and inhibited by sphingomyelin. Recently determined three-dimensional structure of EqtII should enable planning further functional studies that will help us to elucidate its mode of action. In order to investigate the role of tryptophan residues in structure and function of EqtII three single tryptophan fluorescence emission center mutants were produced. Mutants were named according to the position of the remaining tryptophan residue(s)-Trp45, Trp116.117 and Trp149. As determined from FTIR spectroscopy measurements, the tryptophan cluster 112-117 seems to be important for maintaining the structure of EqtII. The emission wavelength maximum in the red region and the high accessibility of Trp149 for acrylamide quenching and NBS modification suggest that this tryptophan residue is solvent exposed. On the contrary, the emission wavelength in the blue region and low accessibility for acrylamide and NBS for mutant Trp116.117 indicate that at least one of the tryptophans at position 116 or 117 is placed in the hydrophobic interior of the protein , thus not exposed to the solvent. The results are in agreement with three-dimensional structure of the toxin. All results indicat that Trp45 is solvent exposed. However, according to the 3D-structure, its side-chain is deeply buried in the hydrophobic core of the protein. We conclude that some conformational changes occurred in the vicinity of this residue. (Abstract truncated at 2000 characters).
Descriptors		CNIDARIAN VENOMS STRUCTURE-ACTIVITY RELATIONSHIP TRYPTOPHAN CYSTEINE CYTOTOXINS CLONING, MOLECULAR SEA ANEMONES POLYMERASE CHAIN REACTION PLASMIDS TRANSFORMATION, GENETIC ESCHERICHIA COLI SPECTROPHOTOMETRY, INFRARED SPECTROMETRY, FLUORESCENCE CHROMATOGRAPHY, HIGH PRESSURE LIQUID