LIPID SPECIFICITY IN THE INTERACTION OF CYTOCHROME-C WITH ANIONIC PHOSPHOLIPID-BILAYERS REVEALED BY SOLID-STATE P-31 NMR

Phosphorus-31 NMR has been used to investigate the interaction of cyto chrome c with bilayers of the anionic lipids dioleoylphosphatidylglyce rol (DOPG), dioleoylphosphatidylserine (DOPS), and diacylphosphatidyli nositol (diacylPI). All P-31 NMR spectra revealed the typical line sha pes characteristic of phospholipids in liquid-crystalline bilayers. Th e effects on the P-31 chemical shift anisotropy (CSA) for each system reflect particular modes of phospholipid headgroup interaction with cy tochrome c. A distinct increase in the CSA for DOPS bilayers was obser ved upon binding of cytochrome c, which is likely to arise from a part ial restriction of the amplitude of motion on this phospholipid headgr oup. P-31 NMR spin-lattice (T-1) relaxation times of the various phosp holipid-cytochrome c complexes show that conformational changes occur in the protein on binding to anionic phospholipids. These protein conf ormational changes are observed through paramagnetic enhancement of th e measured P-31 spin-lattice relaxation times for lipid phosphates. Ho wever, the P-31 T-1 values for the various complexes with cytochrome c show a different temperature dependence for each lipid, revealing dif ferent modes of protein interaction far each of the different lipid he adgroups. The phosphate of DOPS was most efficiently relaxed by cytoch rome c, while the relaxation of the phosphate in the PI headgroup was not affected. The relaxation profile for DOPG-bound cytochrome c shows a more complex behavior, where the lipid phosphorus relaxation is str ongly enhanced above 15 degrees C, but not significantly affected at l ower temperatures. It was found that the enhancement of lipid phosphor us relaxation is a result of the conformational changes in the protein , in which the heme becomes accessible to lipid phosphate upon binding to charged bilayer surfaces. These conclusions from the NMR experimen ts are supported by differential scanning calorimetry results, which i ndicate that the binding of cytochrome c to the anionic phospholipid b ilayers results in a loosening and/or destabilization of the overall p rotein structure. DOPS- and diacylPI-bound cytochrome c shows the larg er destabilization of the protein structure as revealed by the poor co operativity of its thermally induced denaturation, while in the DOPG-c ytochrome c complexes the protein seems to preserve a more nativelike structure.