Avidin-FITC topological studies with three cysteine mutants of equinatoxin II, a sea anemone pore-forming protein

Biochemical and Biophysical Research Communications
G AnderluhP Maček

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 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.

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Citations

Jan 29, 2000·European Journal of Biochemistry·C van KraaijO P Kuipers
Nov 2, 2001·Toxicon : Official Journal of the International Society on Toxinology·Gregor Anderluh, Peter Macek
Oct 27, 2015·Biochimica Et Biophysica Acta·Akiko Yamaji-HasegawaToshihide Kobayashi
Apr 23, 2011·Toxicon : Official Journal of the International Society on Toxinology·A ValleS Schreier
Aug 21, 2014·Chembiochem : a European Journal of Chemical Biology·Matthew A B BakerMark I Wallace
Apr 8, 2020·Biomolecules·Santos Ramírez-CarretoClaudia Rodríguez-Almazán
Dec 20, 2014·The Journal of Biological Chemistry·Yamunadevi SubburajAna J García-Sáez
May 26, 2018·Marine Drugs·Elena LeychenkoEmma Kozlovskaya

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