Spectroscopic Studies of the Interaction of Native and TOAC-Labeled Peptide Hormones with Model Membranes: Angiotensin II

The peptide hormone angiotensin II (DRVYIHPF, AII) plays an important role in the renin-angiotensin-aldosterone system. AII derivatives containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) replacing residues 1 (TOAC1-AII) and 3 (TOAC3-AII) were synthesized and their conformational properties, as well as those of the native peptide, were examined in the presence of model membranes - micelles of 1-palmitoyl-2-hydroxy-phosphatidylcholine (LPC) and 1:1 mol:mol LPC:1-palmitoyl-2-hydroxy-phosphatidylglycerol (LPG) and large unilamellar vesicles (LUV) of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1:1 mol:mol POPC:1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG). Experiments were conducted at pH 4.0, 7.0, and 10.0 to evaluate the effect of peptide charge on peptide-membrane interaction. Fluorescence spectra showed that the peptides bound to negatively charged micelles to a much larger extent than to zwitterionic micelles. CD spectra of AII and TOAC1-AII showed acquisition of secondary structure upon binding to LPC:LPG micelles at pH 4.0; the changes occurred to a lesser extent at the higher pHs. In the case of TOAC3-AII, binding had a small effect on peptide conformation since the TOAC ring imposes a more constrained conformation already in solution. In the case of bilayers, the peptides interacted only with POPC:POPG LUV, especially at pH 4.0. Line broadening of EPR spectra of the labeled peptides also provided evidence for interaction of the labeled peptides with negatively charged micelles and bilayers. In several cases, two-component spectra were obtained, one due to the peptides in solution and the other to the bilayer-bound population, allowing for the calculation of partition coefficients. The rigidity of the TOAC-labeled analogue is very likely responsible for its inability to acquire the correct receptor-bound conformation, leading to loss of biological activity. These data show that spectroscopic studies can provide relevant information regarding peptide-membrane interaction.