A novel hypothalamic neuropeptide of the RFamide family, comprising 26 amino acids residues and thus termed 26RFa, has been recently characterized in human, and was found to be the endogenous ligand for the orphan G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa provokes a robust increase in food intake in rodents. In the present study, we have investigated the solution conformation of 26RFa by using two-dimensional NMR spectroscopy in different media. In water, 26RFa exhibits mainly a random coil conformation although the presence of a nascent helix was detected between residues 6 and 15. In methanol, 26RFa adopts a well-defined conformation consisting of an amphipathic alpha-helical structure (Pro4-Arg17), flanked by two N- and C-terminal disordered regions. The strong conservation, from amphibians to mammals, of the amino acid sequence corresponding to the amphipathic helix and to the C-terminal flexible octapeptide of 26RFa, suggests that these two domains are crucial for the interaction of the peptide with its receptor.

A set of high-field, low-temperature NMR experiments has been conducted on various mixtures of (MeLi)-Li-6 and (LiBr)-Li-6 in toluene. All the Li-6 and H-1 signals of the (MeLi)(n)(LiBr)(4-n) aggregates were unambiguously assigned via one- and two-dimensional (HOESY and COSY) experiments. The influence of the MeLi/LiBr ratio on the concentration of these different aggregates in solution was then studied. The data suggest that the populations of the five possible complexes follow an almost purely statistical distribution with the exception of the MeLi(LiBr)(3) species. The later, which was found to be less abundant than expected, is also less favored on the basis of aggregation energies obtained from density functional theory calculations.

The complexes between methyllithium and chiral 3-aminopyrrolidine (3-AP) lithium amides bearing a second asymmetric center on their lateral amino group were studied using multinuclear ((1)H, (6)Li, (13)C, (15)N) low-temperature NMR spectroscopies in tetrahydrofuran-d(8). The results indicate that lithium chelation forces the pyrrolidine ring of the 3-AP to adopt a norbornyl-like conformation and that robust 1:1 noncovalent complexes between methyllithium and 3-AP lithium amides form in the medium. A set of (1)H-(1)H and (1)H-(6)Li NMR cross-coupling correlations shows that the binding of methyllithium can take place along the "exo" or the "endo" face of this puckered structure, depending on the relative configuration of the lateral chiral group. This aggregation step renders the nitrogen of the 3-amino group chiral, the "exo" and "endo" topologies corresponding to the (S) and (R) configurations, respectively, of this atom. Density functional theory calculations show that the "exo" and "endo" arrangements are, for both diastereomers, almost isoenergetic even when solvent is taken into account. This result suggests that the formation of the mixed aggregates is under strict kinetic control. A relationship between the topology of these complexes and the sense of induction in the enantioselective alkylation of aromatic aldehydes by alkyllithiums is proposed.

The octadecaneuropeptide (ODN; QATVGDVNTDRPGLLDLK) and its C-terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca2+]i) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [d-Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [d-Leu5]OP. On-resin homodetic backbone cyclization of OP yielded an analog, cyclo1-8 OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca2+]i in cultured rat astrocytes. Cyclo1-8 OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover. Conversely, the cyclo1-8 [d-Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca2+]i and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two-dimensional 1H-NMR and molecular dynamics. Cyclo1-8 OP exhibited a single conformation characterized by a gamma turn comprising residues Pro2-Leu4 and a type III beta turn encompassing residues Leu5-Lys8. Cyclo1-8 [d-Leu5]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg-Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs.

In this paper, we demonstrate the utility of 1H–19F chemical shift correlation 2D NMR experiments for the characterization of fluorinated compounds. 1H–19F hetero-COSY provides much more information compared to the used 1H–19F HMQC and HMBC experiments. We performed 1H–19F hetero-COSY experiment and its selective version, both with pulsed field gradients, to obtain resonance assignments for the characterization of fluoromolecules. These experiments are illustrated by spectra of C2F5(VDF)2I, used here as a model compound, and spectra of the fluorinated terpolymer XC2000S containing vinylidenefluoride (VDF), tetrafluoroethylene (TFE) and allyloxypropandiol (AOPD).

Peptide E is a 25-amino acid peptide derived from proenkephalin A that was originally isolated from the bovine adrenal medulla. Bovine peptide E (BPE), which possesses a Met- and a Leu-enkephalin sequence at its N- and C-terminus, respectively, has been described as a highly potent and selective μ-opioid receptor agonist. Paradoxically, the frog counterpart of peptide E (FPE), which exhibits only two amino acid substitutions (Met15 → Gln and Leu25 → Met) compared with BPE, was found to be totally devoid of antinociceptive activity. To decipher this apparent discrepancy, we have decided to compare the structural and pharmacological characteristics of FPE, BPE, and the chimeric peptide [Gln15]BPE (Q15BPE). In methanol, all three peptides exhibited virtually the same conformation, the central region of each peptide (residues 10–20) being involved in a regular helix. Intracerebroventricular administration of FPE, BPE, or Q15BPE, at doses up to 1 000 ng per mouse, did not induce any analgesic effects, as evaluated by the hot plate and writhing tests, whereas, in the same tests, β-endorphin at a dose of 100 ng provoked profound analgesia. Concomitant administration of FPE, BPE, or Q15BPE (100 ng) with the aminopeptidase-N inhibitor bestatin (50 μg) or the endopeptidase 24–11 inhibitor thiorphan (10 μg) did not produce analgesic responses. Antinociceptive effects were only observed when very high doses of FPE, BPE, and Q15BPE (10 000 ng per mouse) were administered. These data clearly demonstrate that, contrary to what has been previously reported, peptide E is virtually devoid of opioid activity.

The solution structure of the R2 repeat of the DNA binding domain of the protooncogene c-Myb contains a N-terminal structural motif comprising two antiparallel helices. The motif is stabilized by interactions involving conserved residues. The recognition region in C-terminal position is flexible. This structure differs from that of R2 of another c-Myb protein.

The three-dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two-dimensional nmr spectroscopy combined with molecular modeling. This 14-residue peptide, Ac Aib1 Asn2 Leu3 Aib4 Pro5 Ala6 Ile7 Aib8 Pro9 Iva10 Leu11 Aib12 Pro13 Leuol14 (Aib, α-aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short-sequence peptaibol class characterized by an acetylated N-terminus, a C-terminal amino alcohol, and the presence of three Aib-L-Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20-residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane-perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage-gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence-specific 1H- and 13C-nmr assignments and conformational nmr parameters (3JNHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty-seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β-turns twisted into a continuous spiral from Asn2 to Leuol14 and forming a 26 Å long helix-like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn2 side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φi = −90°, Ψi = −27°; Φi+1 = −98°, Ψi+1 = −17°; Φi+2 = −49°, Ψi+2 = −50°; Φi+3 = −78°, Ψi+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)n sequences, the (Xaa–Yaa–Aib–Pro)-β-bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 310-helix made of a succession of type III β-turns and from the β-bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999

Synthetic alamethicin analogs, in which all Aib residues had been replaced by Leu (L2) then proline 14 replaced by an alanine (L5), were studied in SDS micelles using circular dichroism and NMR spectroscopy. Nuclear Overhauser effects were used as constraints for molecular modelling. The structures determined for both peptides in SDS micelles were compared with those previously obtained in methanol in order to establish a secondary structure/ionophore activity relationship. Our results indicated that a shortening of peptide helices could be responsible for the observed decrease in ion channel lifetimes. However, the length of helices may not by itself explain the drastic destabilization of channels when Pro14 of alamethicin is replaced by Ala in L5. Indeed analysis of the helical wheel of L5 reveals heterogeneity in the amphipathicity depending on the medium. Thus, loss of amphipathicity seems to underly the observed destabilization of channels. © 1998 European Peptide Society and John Wiley & Sons, Ltd.

JMV635, a nonapeptide analog of the active terminal nonapeptide segment of bombesin, was tested for its ability to stimulate in vitro amylase release from rat pancreatic acinar cells and to inhibit the binding of gastrin-releasing peptide to rat pancreatic acini. It was found to be a full agonist of bombesin and to recognize the bombesin receptor with moderate potency. The NMR proton assignments of JMV635 were achieved, and the conformations of JMV635 in aqueous solution and in trifluoroethanol at 297 K were determined using two-dimensional COSY, HOHAHA, NOESY and ROESY experiments. In trifluoroethanol, JMV635, like the active part of bombesin, showed a partial alpha-helical structure. These results were confirmed by circular dichroism and refined by restrained molecular dynamic methods. Structure calculations, using the distance and angle restraints obtained from NMR data on JMV635, gave a total of 75 structures which could be aligned to a root mean square deviation of the bond length of 0.007 A and of the valence angle of 1.55 degrees for the backbone atoms of the amino acid residues. The conformation is a well-defined right-handed alpha-helix in the C-terminal Q2-G6 segment and is less structured in the three C-terminal residues.

Human immunodeficiency virus type 1 integrase (HIV‐1 IN) which catalyzes viral DNA integration into the host genome of infected cells represents an attractive target for AIDS therapy. We have previously demonstrated the ability of the IN‐(147−175)‐peptide derived from the catalytic core domain of HIV‐1 IN to inhibit the enzyme activity in vitro . IN‐(147−175)‐peptide contains four heptad repeats and displays a high propensity for coiled‐coil formation while its [P159]IN‐(147−175)‐peptide analog (Lys159←Pro in the protein, Lys13←Pro in the peptide) is unable to form a stable coiled‐coil and is devoid of inhibitory activity [Sourgen, F., Maroun, R. G., Frère, V., Bouziane, M., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240 , 765−773]. Now, we report results from an NMR study on IN‐(147−175)‐peptide and [P159]IN‐(147−175)‐peptide as well as on an optimized [E156, A163, A167]IN‐(147−175)‐peptide that is a better inhibitor of IN than IN‐(147−175)‐peptide. While in aqueous solution, IN‐(147−175)‐peptide and [P159]IN‐(147−175)‐peptide display only nascent helical features, [E156, A163, A167]IN‐(147−175)‐peptide exhibits 20 % of helical content. In 20 % trifluoroethanol/80 % H 2 O, the helix content is the highest for [E156, A163, A167]IN‐(147−175)‐peptide (≈70 %) and the lowest for [P159]IN‐(147−175)‐peptide (≈40 %), due to a local helix break caused by the Pro residue. The NHs of residues in the two central helical heptads (a−g) of IN‐(147−175)‐peptide and [E156, A163, A167]IN‐(147−175)‐peptide display a regular periodic variation of their temperature coefficients in 20 % trifluoroethanol. The b, c and f residues on the hydrophilic face of the amphipathic helix show high coefficients reflecting hydrogen bonded NHs, while the a and d residues on the hydrophobic face exhibit low coefficients, near random‐coil values. The particular arrangement of the hydrophobic side‐chains of a and d residues at the coiled‐coil interface reduces the access of trifluoroethanol molecules to their amide groups. The inability of trifluoroethanol molecules to create interactions with the amide C=O groups, these being required to strengthen the intrahelical C=O H‐N hydrogen bonds, is the main cause for observation of heptadic a and d residues with low NH temperature coefficients. Such effects concern mostly the two central helical heptads of IN‐(147−175)‐peptide and [E156, A163, A167]IN‐(147−175)‐peptide implying that these ones are engaged in stable parallel coiled coils. Our results provide a link between the propensity of peptides for helix formation, their coiled‐coil properties and their efficiency to inhibit IN.

From a set of one- and two-dimensional H-1, C-13 and Li-6 NMR experiments, we propose the formation of a well organised 1:1 tight complex between substituted 3-aminopyrrolidine lithium amides and n-butyllithium. This complex is probably at the origin of the stereoselection observed in the asymmetric condensation of n-butyllithium onto aromatic aldehydes.

By H-1 and C-13 NMR spectroscopy, a structural analysis of l-lithio-2-trimethylsiloxyethylene, formed in Et2O solution, has shown a mixture of several solution species at -75 degrees C; this carbanion 1, stable for a long time at -75 degrees C, undergoes a degradation reaction when the temperature increases to afford by-products, all of which have been analysed and their percentages determined for each temperature.

Secretoneurin is a 33-amino-acid polypeptide generated by proteolytic cleavage of secretogranin II at paired dibasic sequences. It has recently been shown that secretoneurin exerts biological activities such as stimulation of dopamine release from striatal neurons and activation of monocyte migration, suggesting that the peptide may modulate both neurotransmission and inflammatory response. In the present study, we have investigated the conformation of synthetic secretoneurin in methanol solution by two-dimensional 1H-NMR, circular dichroism and molecular modeling. Using sequential information, specific assignments have been made for resonances arising from all protons, except for the labile proton of the N-terminal Thr of the peptide. The solution structure of secretoneurin has been determined by distance geometry and restrained molecular dynamics, using distance and dihedral constraints derived from the NMR data. The conformation obtained is composed of two contiguous alpha-helices comprising residues Glu3-Gln8 and Pro11-Gly25. An excellent concordance was observed between these conformational data and prediction with the AGADIR program for the location for the helices in the sequence. These conformational data should help to elucidate the involvement of the tertiary interactions and to design secretoneurin analogs.

Due to the bend introduced by proline 14 in the conformation of alamethicin (AcUPUAUAQUVUGLUPV UUEQFol), the role of this residue was assumed essential in the barrel-stave model for voltage-gated ion channels. Taking advantage of a previous synthetic alamethicin analogue (L2), in which all eight alpha-aminoisobutyric (U) were replaced by leucines (AcLPLALAQLV LGLLPV LLEQFol), another analogue (L5) was synthesized in order to test the effects of proline-14 substitution by an alanine (AcLPLALAQLVLGLLPVLLEQFol). Previous conductance experiments showed that both high voltage dependence and multistate behavior were conserved. In order to complement these functional results, a conformational study of L5 has been undertaken and compared to L2 using CD, high field nmr, and molecular dynamics. Results show that L5 presents a better ordered structure than L2 particularly in the region of the substitution and in the C-terminal part. These results are discussed as regards the previous hypothesis of the nonessential character of helix bending for the gating of voltage-dependent ion channels.

Alamethicin, a 20‐residue peptaibol, induces voltage‐dependent ion channels in lipid bilayers according to the barrel‐stave model. A synthetic analogue (L2) in which all Aib were replaced by Leu shows a conductance behaviour similar to alamethicin, but channel lifetimes are drastically reduced. Among several hypotheses, a different conformation for L2 might be responsible for this phenomenon by increasing the α‐helical content (alamethicin presents some 3.010‐helical parts) and thus decreasing the length of the transmembrane part. A conformational study of L2 was undertaken using FTIR, CD and NMR spectroscopy, and the secondary structure was compared with alamethicin. These techniques showed an enhanced predominant helical structure as compared to alamethicin. Moreover, the NOE pattern showed an exclusively α‐helical conformation, resulting in a smaller length of the L2 peptide. This shortening somewhat impedes the complete crossing of the membrane, and could then explain the reduction of its ion‐channel lifetimes. © Munksgaard 1995.

The H-1, C-13 and Si-29 NMR spectral characteristics of beta-silylated silyl enol ethers were determined. (n)J(Si, H) measurements (2 < n < 4) were achieved from selective polarization transfer experiments with selective decoupling during acquisition. H-1,Si-29 two-dimensional heteronuclear correlation confirmed the positions of the silylated groups in the molecules.

Antipain [(1-carboxy-2-phenylethyl)carbamoyl-L-arginyl-L-valyl-DL-arginal] is a potent inhibitor of papain and related proteases. The assignment of H-1 NMR signals of the interconverting forms in equilibrium in aqueous solution at pH 5.8 were investigated using two-dimensional DQF-COSY, DOUBLE-RELAYED-COSY, HOHAHA and ROESY nuclear magnetic resonance techniques. The assignments of protons of several forms in equilibrium of the D- and L-arginal antipain isomers were determined in aqueous solution at 300 K, and their corresponding stereochemistry is tentatively proposed. Addition of equimolar amounts of papain resulted in spectral changes and chemical shifts which are compatible with enzyme-L-carbinolamine interactions.