The third helix of antennapedia homeodomain pAntp-(43 -58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides. The conformational and associative behaviour of two peptidic vectors pAntp-(43 -58) and [Pro50]pAntp-(43 -58) has been analyzed by different biophysical methods. pAntp-(43 -58) adopts an amphipathic helical structure in 30 % (by vol.) hexafluoroisopropanol, in perfluoro-tert-butanol and in the presence of SDS micelles.CD spectra indicate that the conformation of [Pro50]pAntp-(43 -58) in contrast to pAntp-(43 -58) is independent of the media used. 'H-NMR spectroscopy in SDS micelles or in perfluoro-tert-butanol allows detection of aggregated peptides probably in a ribbon 2, type conformation. These conformations became the predominant structure when Gln50 was replaced by ProSO. Interproton-distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: a-helix and essentially extended structures. Consecutive CHa(i)lCHa(i+ 1) NOES are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with 4 and y/ angles in the p-sheet and y-turn regions. Fluorescence spectroscopy analysis has shown that the indole groups of both peptides penetrate into SDS micelles; both peptides also induce the formation of micelles at very low concentration of SDS (20 pM). Similar interaction was observed with reverse-phase micelles made of bis(2-ethyhexyl) sodium sulfosuccinate and small unilamellar vesicles (SUV) made of a mixture of phosphatidylcholine/phosphatidylserine. 3'P-NMR of vesicles (SUV and large unilamellar vesicles) indicated that the addition of pAntp analogues did not affect the size of phosphatidylcholine/phosphatidylserine vesicles. The addition of pAntp analogues to lipidic dispersions modulates lipid polymorphism in different ways depending on the mixtures of acidic lipids.

The S/T-XI-X,-N-P-X,-X,-Y highly conserved sequence of the seventh transmembrane (TM VII) segment of G-protein-coupled receptors is not present in the photon receptor bacteriorhodopsin TM VII domain. Despite this noticeable discrepancy in sequence, the X-ray structure of bacteriorhodopsin is generally used as the key structure for modelling all G-protein-coupled receptors. Thus, a kinked truns-Pro helix is usually accepted for the TM VII three-dimensional structure of G-proteincoupled receptors, although Asn-Pro dipeptide mainly induces a type UIII /3-turn conformation in both model peptides and proteins. NMR studies in various solvents and molecular calculations were undertaken in order to gain insight into the conformational behaviour of a 15-residue peptide from the tachykinin NK-1 TM VII domain incorporating this common sequence. The low solubility of this membrane-embedded peptide precludes methanol or micellar systems mimicking membrane environment; thus only dimethylsulfoxide (Me,SO) or chlorofonn/Me,SO mixture could be used. We also found that perfluoro-tert-butanol, which has not been previously used for NMR studies, constitutes an excellent alternative solvent for the analysis of hydrophobic peptides. The postulated kinked truns-Pro helix was only present as a minor conformer in Me,SO and an equilibrium between helical and extended structures existed. From NOE data a type I/III ,&structure, centered around Pro9-Ile10, probably stabilized by an Asx turn, may be postulated. Addition of chloroform in Me,SO increased the percentage of folded structures but no preferential conformation could be proposed. In perfluoro-tert-butanoI/CD,OD (9 : 1) the N-and C-terminal regions presented an a-helical structure, and these two domains were linked by a hinge around Asn-Pro with a y-turn for the preceding residue Tyr7 and either a type I/III p-turn around Pro9-IlelO or cxR orientations for these residues, which are both stabilized by an Asx turn. As determined by energy calculations, these structures were equally as stable as the kinked truns-Pro helix and could constitute key structures for analysing the conformational changes and/or the dynamics of TM VII segment induced by the ligand when interacting with the receptor.The apparent similarity in the hydropathicity profiles of G-protein-coupled receptors [ 1 -41 with that of bacteriorhodopsin [5] has led to the use of this integral membrane protein as a starting point for predicting their three-dimensional structures [6-121, even though bacteriorhodopsin is not coupled to G-proteins. The currently accepted proposal for the topography of G-protein-coupled receptors includes seven transmembrane-spanning domains (TM I-VII) joined together by extracellular and cytoplasmic loops, with the Nterminal side in the extracellular cleft and the C-terminal residues in the cytoplasm. This model, which was first deduced Correspondence to S .