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