AVIDIN-FITC TOPOLOGICAL STUDIES WITH 3 CYSTEINE MUTANTS OF EQUINATOXIN-II, A SEA-ANEMONE PORE-FORMING PROTEIN

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, ar ginine 126, and alanine 179. Accessibility of an introduced thiol grou p 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 t o the solvent. Mutants were also biotinylated with biotin maleimide, e nabling coupling with avidin-fluorescein isothiocyanate (avidin-FITC). After binding and insertion of biotinylated toxins into liposomes, av idin-FITC, which is unable to enter intravesicular compartment through toxin-created pores, was used to discriminate intra-or extravesicular ly located thiols. All the mutated residues are found to be located on the outside of the lipid vesicles. The results proved the biotin-avid in system as suitable for topological studies of proteins creating por es in membranes. (C) 1998 Academic Press.