Cardiotoxin II segregates phosphatidylglycerol from mixtures with phosphatidylcholine: (31)P and (2)H 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 ((31)P) and deuterium ((2)H) nuclear magnetic resonance (NMR) spectroscopy. Adding CTX II to 1:1 POPC/POPG mixtures produced a two-component (31)P NMR spectrum, in which the second component had a decreased chemical shift anisotropy. Simultaneously, the (2)H 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 (2)H NMR. Using magic angle spinning (MAS) (31)P NMR spectroscopy, two isotropic resonances were resolved in the absence of CTX II and were assigned to POPG (0.47 ppm) and POPC (-.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.
Carbone
M A
MA
Department of Chemistry and Erindale College, University of Toronto, Ontario, Canada.
Macdonald
P M
PM
D004179:Cobra Cardiotoxin Proteins / Q000737:chemistry*; D004546:Elapid Venoms / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010713:Phosphatidylcholines / Q000737:chemistry*; D010715:Phosphatidylglycerols / Q000737:chemistry*; D012995:Solubility; D013816:Thermodynamics
D004179:Cobra Cardiotoxin Proteins; D004546:Elapid Venoms; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols