Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues

Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. The analogues [TOAC(34)]-pNPY and [Ala(31),TOAC(32)]-pNPY showed a marked selectivity for the Y-5 receptor, while [TOAC(2)]-pNPY maintained a significant binding also to the Y-2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution. Furthermore, we demonstrated that TOAC-labelling is a valuable tool to investigate changes in the backbone conformation and dynamics.