Author/Editor | Anderluh, Gregor; Barlič, Ariana; Križaj, Igor; Menestrina, Gianfranco; Gubenšek, Franc; Maček, Peter | |
Title | Avidin-FITC topological studies with three cysteine mutants of equinatoxin II, a sea anemone pore-forming protein | |
Type | članek | |
Source | Biochem Biophys Res Commun | |
Vol. and No. | Letnik 242 | |
Publication year | 1998 | |
Volume | str. 187-90 | |
Language | eng | |
Abstract | Equinatoxin II (EqtII) is a cysteinless pore-forming protein from sea anemone Actinia equina. Three cysteine mutants were produced in an E. coli expression system in order to study the topology of lysine 77, arginine 126, and alanine 179. Accessibility of an introduced thiol group in the water soluble mutants was studied by using the thiol specific reagent fluorescein maleimide. In aqueous solution all three mutants were readily modified with the probe, indicating their accessibility to the solvent. Mutants were also biotinylated with biotin maleimide, enabling coupling with avidin-fluorescein isothiocyanate (avidin-FITC). After binding and insertion of biotinyaled toxins into liposomes, avidin-FITC, which is unable to enter intravesicular compartment through toxin-created pores, was used to discriminate intraor extravesicularly located thiols. All the mutated residues are found to be located on the outside of the lipid vesicles. The results proved the biotin-avidin system as suitable for topological studies of proteins creating pores in membranes. | |
Descriptors | CNIDARIAN VENOMS SEA ANEMONES ION CHANNELS CYSTEINE AVIDIN FLUORESCENT DYES DIMERIZATION FLUORESCEIN-5-ISOTHIOCYANATE PROTEIN CONFORMATION MUTAGENESIS |