ACTH BINDING TO PHOSPHOLIPID-BILAYERS - A H-1-NMR STUDY OF THE 5-14, 1-14, AND 1-24 DERIVATIVES

The interaction of the 5-14, 1-14, and 1-24 fragments of ACTH with son icated phospholipid bilayers containing egg yolk phosphatidylcholine ( EPC) either pure or mixed with 10 mole % phosphatidic acid (EPA), was investigated by proton nuclear magnetic resonance (H-1-nmr). The effec ts observed with zwitterionic EPC vesicles were small, indicating a lo w binding of the ACTH derivatives. The N-terminal aromatic resonances of the ACTH peptides were markedly broadened in the presence of negati vely charged vesicles (EPC/EPA 9:1 M/M), while those of the C-terminal end were barely affected showing that ACTH interacts with its N-termi nal fragment. The choline resonance of the EPC molecules of the outer monolayer was shifted and broadened upon ACTH binding to the lipid ves icles, while that of the inner layer was not affected suggesting that the peptide molecules interact only with the external leaflet of the l ipid bilayer The (CH)-H-2 and (CH)-H-4 resonances of the histidine-6 s ide chain were both shifted downfield upon peptide binding to the nega tively charged lipid interface. In the case of the 1-24 derivative, th ese resonances were also split into two signals reflecting two differe nt species of membrane-bound ACTH 1-24. Analysis of the line width and chemical shift variations of the ACTH and lipid resonances observed u pon peptide binding shows that the membrane-binding potency of the sho rter 5-14(+1) fragment, which presents a +1 net charge, is roughly sim ilar to that of the highly cationic 1-24(+6) (net charge +6) derivativ e, implying that the 15-24(+5) segment is not essential for membrane b inding. The nmr measurements at a fixed lipid-to-peptide ratio in the presence of increasing amounts of spin-labeled lipids demonstrate that the N-terminal fragment of ACTH does nor penetrate the hydrophobic co re of the bilayer, and should lie parallel to the membrane surface. (C ) 1997 John Wiley & Sons, Inc.