Avidin-FITC topological studies with three 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, 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 biotinylated toxins into liposomes, avidin-FITC, which is unable to enter intravesicular compartment through toxin-created pores, was used to discriminate intra- or 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.
Anderluh
G
G
Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia. gregor.anderluh@uni-lj.si
Barlic
A
A
Krizaj
I
I
Menestrina
G
G
Gubensĕk
F
F
Macek
P
P
D000818:Animals; D001360:Avidin / Q000031:analogs & derivatives; D019921:Biotinylation; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D011487:Protein Conformation; D012615:Sea Anemones / Q000737:chemistry*
D003064:Cnidarian Venoms; D005456:Fluorescent Dyes; D007473:Ion Channels; C036963:fluorescein isothiocyante avidin; D001360:Avidin; C012307:equinatoxin; D016650:Fluorescein-5-isothiocyanate; D003545:Cysteine