Cardiotoxin II segregates phosphatidylglycerol from mixtures with phosphatidylcholine: P-31 and H-2 NMR spectroscopic evidence

The interaction of the cationic protein cardiotoxin II (CTX II) with mixtures of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was investigated using phosphorus (P-31) and deuterium (H-2) nuclear magnetic resonance (NMR) spectroscopy. Adding CTX II to 1:1 POPC/POPG mixtures produced a two-component P-31 NMR spectrum, in which the second component had a decreased chemical shift anisotropy. Simultaneously, the H-2 NMR quadrupolar splitting measured from both POPC-alpha-d(2) and POPC-beta-d(2) decreased. Thus, CTX II produces an altered macroscopic phase state of the lipid bilayers, and this obscures any effects on bilayer surface electrostatics observed by H-2 NMR. Using magic angle spinning (MAS) P-31 NMR spectroscopy, two isotropic resonances were resolved in the absence of CTX II and were assigned to POPG (0.47 ppm) and POPC (-0.58 ppm). Adding CTX II produced two new isotropic resonances shifted approximately 0.5 ppm downfield. Quantifying the intensities of the various resonance lines revealed that the binding isotherms for different POPC/POPG mixtures shifted onto a universal curve when expressed as a function of the CTX II/POPG ratio. The results indicate that CTX II binds preferentially to POPG and is able to laterally segregate POPG from POPC. Fitting of the binding isotherms was achieved using a two-site model derived from statistical-thermodynamic considerations. One class of binding site is specific for POPG and the other is nonspecific, capable of binding both POPC and POPG.