ABSTRACT
The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7x10(8) M(-1)). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.
Subject(s)
Calorimetry/methods , Cnidarian Venoms/chemistry , Cnidarian Venoms/genetics , Lipids/chemistry , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/genetics , Protein Conformation , Amino Acid Sequence , Animals , Cnidarian Venoms/metabolism , Hemolysis , Models, Molecular , Mutagenesis, Site-Directed , Pore Forming Cytotoxic Proteins/metabolism , Protein Binding , Sea Anemones/chemistry , ThermodynamicsABSTRACT
Wild-type actinoporins StnI and StnII from the sea anemone Stichodactyla helianthus, as well as their NH(2)-terminal six-His tagged versions, have been overproduced in Escherichia coli. Overproduction of both wild-type proteins was only possible after introducing silent mutations within the 5'-end of their original cDNA sequences. These mutations would prevent the formation of RNA secondary structures blocking the ribosome-binding site and the initiation codon. The four recombinant proteins were purified to homogeneity in milligrams amount and characterized from spectroscopic and functional points of view. All the isolated proteins behaved as the corresponding natural ones although the six-His tagged variants exhibited a decreased lytic activity. The strategy described will be useful to allow the production of mutant variants of these proteins and probably of other actinoporins.
