title: Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues.
abstract: Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. 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. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 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 as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.
authors.affiliation: Institute of Biochemistry, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bettio
authors.forename: Volker
authors.initials: V
authors.lastname: Gutewort
authors.forename: Andreas
authors.initials: A
authors.lastname: Pöppl
authors.forename: Michaela C
authors.initials: MC
authors.lastname: Dinger
authors.forename: Olaf
authors.initials: O
authors.lastname: Zschörnig
authors.forename: Arnold
authors.initials: A
authors.lastname: Klaus
authors.forename: Claudio
authors.initials: C
authors.lastname: Toniolo
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003497:Cyclic N-Oxides / Q000737:chemistry*; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017476:Receptors, Neuropeptide Y / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013113:Spin Labels
chemical_list: D003497:Cyclic N-Oxides; D008081:Liposomes; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D013113:Spin Labels; C037109:2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylic acid