Localization of hydrophobic ions in phospholipid bilayers using 1H nuclear Overhauser effect spectroscopy

The binding location for the hydrophobic ions tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-) was studied in sonicated phosphatidylcholine (PC) vesicles by measuring time-dependent and steady-state intermolecular 1H nuclear Overhauser effects (NOE's). Intermolecular cross-relaxation was also investigated by two-dimensional NOE spectroscopy. Information on the distance and order parameter dependence of the NOE's was obtained from a simple simulation of the NOE's in the alkyl chain region. Taken together, the NOE data and the simulation provide strong evidence that TPB- and TPP+, at low concentrations (less than or equal to 10 mol%), are localized in the alkyl chain region of the bilayer. At these lower concentrations of TPP+ or TPB-, no significant effect on lipid 13C T1 or T2 relaxation rates is detected. The proposed location is consistent with the expected free energy profiles for hydrophobic ions and with the carbonyl oxygens or interfacial water as the source of the membrane dipole potential. At higher ion/lipid ratios (greater than or equal to 20 mol%), TPB-/lipid NOE's increase. This results from a specific association of TPB- with the choline head group.