Interaction of the pore-forming protein equinatoxin II with model lipid membranes: A calorimetric and spectroscopic study.
The interactions of equinatoxin II (EqTxII) with zwitterionic (DPPC) and anionic (DPPG) phospholipids and an equimolar mixture of the two phospholipids (DPPC/DPPG) have been investigated by differential scanning calorimetry (DSC), CD-spectropolarimetry, intrinsic emission fluorescence spectroscopy, and ultrasonic velocimetry. EqTxII binds to small unilamellar vesicles formed from negatively charged DPPG lipids, causing a marked reduction in the cooperativity and enthalpy of their gel/liquid-crystalline phase transition. This transition is completely abolished at a lipid-to-protein ratio, L/P, of 10. For the mixed DPPC/DPPG vesicles, a 2-fold greater lipid-to-protein ratio (L/P = 20) is required to abolish the phase transition, which corresponds to the same negative charge (-10) of lipid molecules per EqTxII molecule. The disappearance of the phase transition of the lipids apparently corresponds to the precipitation of the lipid-protein complex, as suggested by our sound velocity measurements. Based on the far-UV CD spectra, EqTxII undergoes two structural transitions in the presence of negatively charged vesicles (DPPG). The first transition coincides with the gel/liquid-crystalline phase transition of the lipids, which suggests that the liquid-crystalline form of negatively charged lipids triggers structural changes in EqTxII. The second transition involves the formation of alpha-helical structure. Based on these observations, we propose that, in addition to electrostatic interactions, hydrophobic interactions play an important role in EqTxII-membrane association.
Poklar
N
N
Department of Chemistry and Biochemistry, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia. natasa.poklar@uni-lj.si
Fritz
J
J
Macek
P
P
Vesnaver
G
G
Chalikian
T V
TV
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003064:Cnidarian Venoms / Q000378:metabolism*; D003603:Cytotoxins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000378:metabolism; D011487:Protein Conformation; D012615:Sea Anemones; D013050:Spectrometry, Fluorescence
D003064:Cnidarian Venoms; D003603:Cytotoxins; D008563:Membrane Lipids; D010715:Phosphatidylglycerols; D015060:1,2-Dipalmitoylphosphatidylcholine; C012307:equinatoxin; C030345:1,2-dipalmitoylphosphatidylglycerol