Author/Editor     Anderluh, G; Barlič, A; Potrich, C; Maček, P; Menestrina, G
Title     Lysine 77 is a key residue in aggregation of eguinatoxin II, a pore-forming toxin from sea anemone Actinia equina
Type     članek
Source     J Membr Biol
Vol. and No.     Letnik 173
Publication year     2000
Volume     str. 47-55
Language     eng
Abstract     Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 - Cys influences the normal development of the aggregate which is required for assembling the functional pore.
Descriptors     CNIDARIAN VENOMS
CYTOTOXINS
ERYTHROCYTE MEMBRANE
LYSINE
SEA ANEMONES
AMINO ACID SEQUENCE
BINDING SITES
ERYTHROCYTES
ESCHERICHIA COLI
HEMOLYSIS
MUTAGENESIS, SITE-DIRECTED
POINT MUTATION
RECOMBINANT PROTEINS
SPECTROSCOPY, FOURIER TRANSFORM INFRARED