title: Salt-induced oligomerization of partially folded intermediates of equinatoxin II.
abstract: Equinatoxin II (EqTxII) is a cytolytic, water-soluble protein which binds to and forms cation-selective pores in lipid membranes. To characterize the native and denatured states of EqTxII and to elucidate the biological role of its oligomers, we have studied salt-dependent heat-induced conformational transitions of EqTxII. To this end, we have employed a variety of experimental techniques, including differential scanning calorimetry, circular dichroism and light absorption spectroscopy, ultrasonic velocimetry, electron microscopy, PAGE, and a hemolytic activity assay. This experimental combination has enabled us to monitor and structurally and thermodynamically characterize temperature-induced conformational transitions and oligomerization of EqTxII at different concentrations of NaCl. At pH 3.0 and 25 degrees C, EqTxII retains its native conformation and remains hemolytically active over a broad range of NaCl concentrations. However, an increase in the salt concentration results in a diminution of the thermal stability of EqTxII. Specifically, the calorimetrically determined denaturation temperature, T(d), and enthalpy, DeltaH(cal), of the toxin decrease with an increase in the salt concentration. Our CD data suggest that the heat-induced denatured state of EqTxII lacks rigid tertiary structure while exhibiting well-defined secondary structure. The amount of the induced, non-native secondary structure of EqTxII depends on the solution ionic strength, temperature, time of incubation at an elevated temperature, and protein concentration. Our combined results suggest that, in the presence of salt, an increase in temperature results in formation of the partially unfolded state of the toxin that oligomerizes and forms biologically inactive, water-soluble aggregates.
authors.affiliation: Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. natasa.poklar@uni-lj.si
authors.forename: Natasa Poklar
authors.initials: NP
authors.lastname: Ulrih
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Peter
authors.initials: P
authors.lastname: Macek
authors.forename: Tigran V
authors.initials: TV
authors.lastname: Chalikian
mesh_terms: D001704:Biopolymers / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003064:Cnidarian Venoms / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D012965:Sodium Chloride / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001704:Biopolymers; D003064:Cnidarian Venoms; D012965:Sodium Chloride; C012307:equinatoxin