Peptides 26 (2005) 1825–1834 Peptide–lipid interaction monitored by spin labeled biologically active melanocortin peptides Roberto M. Fernandez a , Renata F.F. Vieira b , Cl ´ ovis R. Nakaie b , Amando S. Ito c , M. Teresa Lamy a,∗ a Instituto de F´ ısica, Universidade de S˜ ao Paulo, CP 66318, CEP 05315-970, S˜ ao Paulo, SP, Brazil b Departamento de Biof´ ısica, Universidade Federal de S˜ ao Paulo, S˜ ao Paulo, SP, Brazil c Faculdade de Filosofia Ciˆ encias e Letras de Ribeir˜ ao Preto, Universidade de S˜ ao Paulo, Ribeir˜ ao Preto, SP, Brazil Received 30 July 2004; accepted 7 December 2004 Available online 11 July 2005 Abstract The present work comparatively analyzes the interaction of -MSH and its more potent and long-acting analog [Nle 4 , D-Phe 7 ]-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac 0 and the time resolved fluorescence of Trp 9 present in the peptides. The Toac 0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pK a 7.5, possibly that of His 6 , can be clearly monitored by peptide–lipid partition. Trp 9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp 9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac 0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone. © 2005 Elsevier Inc. All rights reserved. Keywords: Melanocortins; Toac; ESR; -MSH; NDP-MSH; Bilayer-partition; pH titration; Trp; Time resolved fluorescence 1. Introduction Melanocortin peptides are known to interact with mem- brane proteins belonging to the super family of receptors coupled to the G-protein, which stimulate the adenosine cyclic 3 ′ ,5 ′ -phosphate (cAMP) signal transduction pathway [11]. However, the lipid phase of the cell membrane possibly plays an active role in the peptide–receptor interaction, not only by increasing the peptide concentration at its surface, by electrostatic effects (anionic lipids/cationic peptide), but also by changing the peptide structural conformation, making it adjustable to the receptor. Besides, the peptide–lipid partition ∗ Corresponding author. Tel.: +55 11 3091 6829; fax: +55 11 3813 4334. E-mail address: mtlamy@if.usp.br (M.T. Lamy). would make the peptide more accessible to the membrane protein receptor, and/or the presence of the peptide inside the bilayer could change the bilayer structure favoring an appro- priate receptor conformation. Considering the possible biological relevance of the lipid phase, -MSH and several active analogs have been tested for lipid affinity, membrane depth penetra- tion and peptide/bilayer structural alterations. Using lipids spin-labeled at different chain positions, incorporated in dimyristoyl phosphatidylglycerol (DMPG) bilayers, we have shown that the two cationic peptides, the hormone - MSH (Ac-Ser 1 -Tyr 2 -Ser 3 -Met 4 -Glu 5 -His 6 -Phe 7 -Arg 8 -Trp 9 - Gly 10 -Lys 11 -Pro 12 -Val 13 -NH 2 ) and the biologically more potent analog [Nle 4 , D-Phe 7 ]-MSH (NDP-MSH), inter- act with anionic DMPG bilayers, changing the membrane 0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.12.030