Conformational analogy between substance P and physalaemin.

The three-dimensional structure of physalaemin, pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2, has been studied by one- and two-dimensional 500 MHz NMR spectroscopies in two solvents: methanol and dimethyl sulfoxide. As previously observed for substance P in methanol, the core of physalaemin 4----8 is folded into an helical conformation. This structure is stabilized by the presence of a salt bridge between Asp-3 and Lys-6 in both solvents. The only differences observed reside in the N-terminal sequences; the N-terminal tripeptide of substance is flexible whereas that of physalaemin is in an extended conformation.

Binding of substance P to monolayers and vesicles made of phosphatidylcholine and/or phosphatidylserine.

Analyses of interactions between substance P (SP) and phospholipids were performed by combined surface pressure and surface potential measurements in monolayers and by 13C-NMR experiments on liposomes. This study was carried out using synthetic SP molecules: [1-13C-Gly9]SP and [1-13C-Gly2]SP. Injection of SP into the aqueous subphase led to an expansion of phosphatidylcholine (PtdCho) or phosphatidylserine (PtdSer) monolayer surface area. An apparent association constant of SP for PtdSer was estimated to be around 10(6)-10(-7) M-1. The surface potential delta V/n varied linearly with the molecular area whereas the variation of surface pressure was biphasic, suggesting that at least two binding states contributed to the monolayer expansion. These two states Si (SP is inserted into the bilayer) and Ss (SP is stuck on the surface) were observed on vesicular membranes by 13C-NMR. The kinetic of interconversion between these two states can be estimated by NMR, the Ss state being the stablest one. No perpendicular insertion of SP into these vesicular preparations seemed to occur, as previously postulated. However, SP might form aggregates in contact with these model systems, leading to a loss of permeability of the lipid vesicles.

Analysis of tachykinin-binding site interactions using NMR and energy calculation data of potent cyclic analogues of substance P.

The three-dimensional structures of [Cys3,6,Tyr8]-, [Gly2,Cys3,6,Tyr8]- and [DCys3,Cys6]substance P, designed as conformational analogues of substance P, have been studied by 1H-NMR (500 MHz) in different solvents and by energy calculations. As previously observed for substance P and physalaemin, two tachykinins acting via the NK-1 receptor, [Cys3,6,Tyr8]substance P presents an alpha-helical structure of the 4----8 sequence in methanol. This structure is stabilized by a beta-turn III via the formation of three hydrogen bonds involving the Cys-6, Phe-7 and Tyr-8 NH groups. In contrast to substance P, two of these hydrogen bonds are still present in dimethyl sulfoxide and in water the Cys-6 NH hydrogen bond is the only one remaining, such that a beta-turn structure inside the ring can be envisaged. In close agreement with the NMR data, the energy calculations lead to three types of folding for the core of [Cys3,6,Tyr8]substance P: a beta-turn III, a less stable beta-turn I (delta E = 3 kcal), and a beta-turn II (delta E = 4.6 kcal). The structure of Gly-Leu-Met-NH2 is strongly affected by changing the hydrophobicity of the medium. The most stable calculated conformation is the helix; however, numerous unrelated structures are destabilized by about 2-3 kcal/mol. These data are analyzed and discussed in connection with the high potency of [Cys3,6,Tyr8]substance P for both the NK-1 and NK-3 binding sites; that is the internal region of tachykinins (non-homologous amino acids) might present a similar three-dimensional structure when bound to the receptors (which may be at the origin of some lack of selectivity), whereas paradoxically the selectivity may be due to the common C-terminal sequence.

New total synthesis and high resolution 1H NMR spectrum of platelet-activating factor, its enantiomer and racemic mixtures.

A new method of synthesis of octadecyl platelet-activating factor (PAF; 1-O-octadecyl 2-O-acetyl sn-glycero-3-phosphorylcholine) is described. Its advantage is to proceed by way of the 'lyso-PAF' which may be substituted by various groups, while avoiding the inconveniences of the total synthesis already described. Moreover, the intermediates in synthesis are easier to purify, with better yields. The platelet-aggregating activity of synthetic PAF, is enantiomer (3-O-octadecyl) and racemic mixtures were 2.7 . 10(-10), 1.7 . 10(-7) and 2.2 . 10(-10) M, respectively. These results indicate the stereospecificity of platelet activation induced by PAF.

The fine structure of two DNA dodecamers containing the cAMP responsive element sequence and its inverse. Nuclear magnetic resonance and molecular simulation studies.

1H and 31P n.m.r. (nuclear magnetic resonance) spectroscopy have been used in conjunction with molecular simulation to determine the structure of two DNA dodecamers. The first of these, CATGACGTCATG, contains the octameric sequence CRE (cAMP responsive element), while the second is the reversed sequence, GTACTGCAGTAC. Structure determination was based on both NOESY (nuclear Overhauser spectroscopy) derived distances and COSY (correlated spectroscopy) dihedral angle data. Access to the 31P spectra also allowed the epsilon backbone angles to be determined. Considerable care was taken in deriving structural parameters from the n.m.r. data and an excellent level of agreement is obtained with the simulated conformations. Both dodecamers are found to belong to the B-DNA family; however, there is a striking difference between the CRE sequence and its inverse, the former conformation alone showing a strong structural heterogeneity.

Structural deviations at CpG provide a plausible explanation for the high frequency of mutation at this site. Phosphorus nuclear magnetic resonance and circular dichroism studies.

CpG sites in DNA are hotspots for mutations leading to human genetic disorders. However, the structural basis for these events were still unclear and necessitated a deeper evaluation. Our experiments with phosphorus-31 nuclear magnetic resonance, ultraviolet-melting and circular dichroism on two related CpG-containing octanucleotide duplexes show that CpG is a malleable step whose conformation and thermal stability are strongly dependent on the nature of its flanking steps. We conclude that the CpG step may exert a deleterious structural influence on the helix very much like the mismatch containing steps. This peculiar property of CpG should constitute a molecular basis for its recognition by various ligands as well as for mutations affecting CpG and hence an explanation for its rarity in vertebrate genomes.

Investigation of the regio- and stereo-selectivity of deoxyguanosine linkage to deuterated 2-hydroxyestradiol by using liquid chromatography/ESI-ion trap mass spectrometry.

From previous studies on the reactivity of estradiol 2,3-quinone towards deoxyribonucleosides, it was demonstrated that several isomeric adducts were formed. Although adduction on steroid ring A or B has been evidenced using sequential MS(n) experiments, in some cases attachment positions are difficult to identify unambiguously. In this work, 2-hydroxyestradiol labeled with deuterium at various positions [6beta (1); 6alpha-7alpha (2); 6alpha-6beta-7alpha (3)] have been used. Isomeric adduct differentiation could be achieved using LC-ESI-MS(n). The m/z shift of the quasi-molecular ions as well as the fragmentation pathways suggested that adduction could occur on both C6 and C9 sites of the steroid B ring: Nucleophilic attack of the base on the C6 position of the steroid led to major adducts and addition of the base on the activated C9 site gave minor adducts that were found to be unstable. LC-MS(n) experiments carried out under deuterated medium provided information about some fragmentation processes by studying the m/z shift of fragment ions: (1) the loss of deoxyribose from the quasi-molecular ions took place according to a process involving a deuterium transfer from the deoxyribose alcohol function; (2) the cleavage of the steroid-base linkage involved a deuterium transfer from the hydroxy group of the catechol and likely occurred via the formation of an ion-dipole complex. The model studies conducted in this work provide new information on the fragmentation mechanisms of covalent adducts formed from estrogen quinones and deoxyguanosine, the most reactive DNA base. Besides, the first unequivocal characterization of adducts involving the steroid C9 position is shown by using deuterium labeled estrogen quinones.

Influence of the replacement of amino acid by its D-enantiomer in the sequence of substance P. 2. Conformational analysis by NMR and energy calculations.

The D-enantiomer of residues 2, 4, 5, 6, 7, 8, 10 and 11 was introduced in the sequence of Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH1. The achiral glycine was replaced by a D-Ala residue. The conformations of the D-substituted analogues were analysed by NMR and molecular energy calculations. Introduction of a D-amino acid in the address sequence of SP (1 to 5) distorted the helical structure of [D-Pro2]SP and [D-Pro4]SP. A D-glutamine in position 5 hampered the formation of an helix, the core of [D-Gln5]SP was stabilized by the presence of two beta-turns. The exact fitting of both Phe7 and Phe8 in the binding pocket can be achieved by either an alpha- or a 3(10) helix or two beta-turns types I and II'. Replacement of an amino acid in the message sequence, 6 to 11, drastically decreased the potencies of the corresponding analogues, different conformational modifications were observed. [D-Gln6]SP presented two beta-turns, however, the types of beta-turns should orientate the side-chains in such a way that [D-Gln6]SP did not fit in the binding site. The conformations of [D-Phe7]SP and [D-Phe8]SP were completely changed, a more or less extended structure being observed. Modifications in the Gly-Leu-Met-NH2 sequence did not affect the helical structure of the core of [D-Ala9]SP, [D-Leu10]SP and [D-Met11]SP, but decreased the percentage of extended structure of the C-terminal tripeptide.

An NMR study of d(CTACTGCTTTAG).d(CTAAAGCAGTAG) showing hydration water molecules in the minor groove of a TpA step.

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-H1'(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment (approximately 3.9A) and a sudden widening at the T10:A15 base-pair (approximately 5.3A), the standard B-DNA distance being approximately 5A. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n> or =2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for A11 ending the 5'TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the A11:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to A11 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.

Selective agonists of tachykinin binding sites.

Three types of binding sites for the mammalian tachykinins, ie Substance P (SP) Neurokinin A (NKA) and Neurokinin B (NKB), have been found in both the central and peripheral nervous systems. Substance P binds to the NK-1 subclass of binding site while NKA and NKB are less selective endogenous ligands, which preferentially interact with the NK-2 and NK-3 subclasses of binding sites, respectively. Complementary strategies, including 3-dimensional structure analysis by NMR spectroscopy and structure-activity relationship led to the design of selective agonists of these binding sites. [Pro9] SP, [Pro10] SP and the cyclic analogues [Cys3,6, Tyr8, Pro9] SP and [Cys3,6, Tyr8, Pro10] SP are selective NK-1 agonists. [Lys5] NKA(4-10) is a water soluble NK-2 potent agonist. Finally, [Pro7] NKB, which completely discriminates NK-2 and NK-3 binding sites, is a water-soluble NK-3 selective agonist.

Chemical structure of a prebiotic analog of adenosine.

Upon heating a dry mixture of ribose and adenine, condensation products are formed. They were identified as isomers of N6-ribosyl-adenine (Fuller, Sanchez and Orgel, 1972). Due to the current interest in nucleotide analogs as potential constituents of primitive RNA catalysts, the products were further characterized by mass spectroscopy and proton NMR. Our results fully substantiate the previous proposals.

Preferential conformation of substance P in solution.

The three-dimensional structure of substance P has been studied by 1H-NMR, (500 MHz), and by circular dichroism (CD) in different solvents. The analysis of the different NMR parameters suggest that substance P adopts a rather extended structure in dimethylsulfoxide and pyridine. In water, besides the aggregation phenomenon, the monomeric substance P presents a complex conformational equilibrium. The addition of sodium dodecylsulfate to the aqueous solution induces, as shown by CD spectroscopy, a preferential alpha-helical conformation. And in methanol three structural conclusions may be drawn: the flexibility of the N-terminal Arg-Pro-Lys, the alpha-helical structure of Pro4-Gln5-Gln6-Phe7-Phe8 and the interaction of the C-terminal carboxamide with the primary amides from both glutamines.

Drug-DNA interactions: spectroscopic and footprinting studies of site and sequence specificity of elliptinium.

The binding of the antitumoral ellipticine derivative 2-methyl-9-hydroxyellipticinium acetate (elliptinium; NMHE) to DNA was analyzed by the combined use of DNase I footprinting and spectroscopic methods. Using two fragments of pBR322 DNA, five discrete NMHE binding sites of 5-7 protected base pairs (bp) were detected by footprinting at 4 degrees C on the analyzed regions. These corresponded to alternating pyrimidines and purines. The inactive derivative 2-methyl ellipticinium acetate L(NME) lacking a hydroxy group failed to demonstrate DNA protection even at low temperature. Ultraviolet-absorption and 1H-nmr analysis was performed using two autocomplementary octanucleotides d(TGACGTCA) (I) and d(ACTGCAGT) (II). The uv-absorption titrations resulted in an intercalative binding mode for NMHE in the oligomers. Analysis of the derived biphasic Scatchard plots yielded two binding sites corresponding to approximately 6-bp and 2-bp sizes and characterized by apparent association constants K1 approximately 10(8) M-1 and K2 approximately 10(6) M-1, respectively. The 1H-nmr analysis of exchangeable (imino) protons and nonexchangeable protons performed in the one- and two-dimensional modes confirmed the intercalation of NMHE, and further revealed the existence of multiple sites on DNA. Assuming that imino resonance line width concerned the sole kinetic effects, 10-ms order lifetimes were estimated for the drug-oligonucleotide complexes at 7 degrees C, pH 7, and 0.1 ionic strength. Finally, examination of every drug-DNA spectra in the light of the footprinting results indicated that there was a preference for binding of NMHE to the CpG (octamer I) and TpG (octamers I and II) steps.

Conformational and associative behaviours of the third helix of antennapedia homeodomain in membrane-mimetic environments.

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. 1H-NMR spectroscopy in SDS micelles or in perfluoro-tert-butanol allows detection of aggregated peptides probably in a ribbon 2(7) type conformation. These conformations became the predominant structure when Gln50 was replaced by Pro50. Interproton-distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: alpha-helix and essentially extended structures. Consecutive CH alpha (i)/ CH alpha (i + 1) NOEs are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with phi and psi angles in the beta-sheet and gamma-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 microM). 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. 31P-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.

Three-dimensional structure of the highly conserved seventh transmembrane domain of G-protein-coupled receptors.

The S/T-X1-X2-N-P-X3-X4-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 trans Pro-helix is usually accepted for the TM VII three-dimensional structure of G-protein-coupled receptors, although Asn-Pro dipeptide mainly induces a type I/III beta-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 (Me2SO) or chloroform/Me2SO 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 trans-Pro helix was only present as a minor conformer in Me2SO and an equilibrium between helical and extended structures existed. From NOE data a type I/III beta-structure, centered around Pro9-Ile10, probably stabilized by an Asx turn, may be postulated. Addition of chloroform in Me2SO increased the percentage of folded structures but no preferential conformation could be proposed. In perfluoro-tert-butanol/CD3OD (9:1) the N- and C-terminal regions presented an alpha-helical structure, and these two domains were linked by a hinge around Asn-Pro with a gamma-turn for the preceding residue Tyr7 and either a type I/III beta-turn around Pro9-Ile10 or alpha R 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 trans-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.

Calpha methylation in molecular recognition. Application to substance P and the two neurokinin-1 receptor binding sites.

Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Calpha methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Calpha methylated analogues of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Calpha methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Calpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [alphaMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Calpha methylation of SP are sequence dependent. Moreover, a single Calpha methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha-aminoisobutyric acid. Furthermore, Calpha methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other potent SP analogues.

Topographic analysis of the S7 binding subsite of the tachykinin neurokinin-1 receptor.

Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2-for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S. 3R)- and (2S, 3S)-indanylglycines, E- and Z-alpha, beta-dehydrophenylalanines, and 2(S)-alpha, beta-cyclopropylphenylalanines [delta E Phe. delta Z Phe. inverted delta E2 (S) Phe, and inverted delta Z 2 (S) Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (delta E Phe, delta Z Phe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (-) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is small since the other rotamer can be detected in [(2S, 3R)Ing7]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe7 substituted analogues of SP must come from steric hindrance in the S7 binding site, which drastically restricts the space filling around the C alpha-C beta bond of residue 7.

Behaviour studies of the fungicide cymoxanil in two strains of the fungus Botrytis cinerea and in haemolymph of locust and lobster. I. In situ monitoring by internal surface reversed-phase high-performance liquid chromatography.

A method for following the metabolism of the fungicide cymoxanil in various biological media is described. By using a recently developed high-performance liquid chromatographic method, with an internal surface reversed-phase column, it is unnecessary to clean up the sample before analysis. Thus this technique makes monitoring in fungi as well as in arthropod haemolymph easier and faster.

Effect of distortions in the phosphate backbone conformation of six related octanucleotide duplexes on CD and 31P NMR spectra.

We examined the structural properties of six octanucleotide duplexes d(TGACGTCA), d(ACTGCAGT), d(CTTCGAAG), d(CATCGATG), d(GTACGTAC), and d(CATGCATG). Circular dichroism (CD) and 2D 31P and 1H NMR spectroscopies were used in conjunction. Although of the B-DNA type, it was possible to arrange CD spectra into two families, A and B. Family A resembled poly(dG-dC) with a positive signal at approximately 280 nm and a negative one at approximately 260 nm, while family B resembled poly(dA-dT) with a positive signal at approximately 270 nm and a negative one at approximately 250 nm. All 31P resonances were assigned through constant-time heteronuclear 31P-1H correlated spectra. J(H3'-P) coupling constants related to dihedral angeles epsilon (C4'-C3'-O3'-P) were determined from 1H-31P J-resolved selective proton-flip 2D experiments. A good correlation was observed between 31P chemical shifts and coupling constants for all oligonucleotides. The patterns of these two parameters vs the base position along the sequences were almost similar. They were confronted with CD spectra. The results indicated that the position and magnitude of the signals were mainly affected by the CpG and ApT steps whose 31P chemical shifts were the farthest away from the mean 31P chemical shift value. This is in keeping with greater rigidity at these steps and should explain the influence of the local order on the shape of the CD spectra. Lastly, both UV absorption and 31P chemical shifts vs temperature provided normal temperature melting (Tm) values for all of the octanucleotide duplexes except for d(CTTCGAAG), for which the Tm was approximately 10 degrees C lower compared to its counterpart d(CATCGATG). The decrease in the thermal stability of this octanucleotide duplex was imputed to its contained TT and AA repeats, which might be able to induce correlated base destacking and phosphate group distortion in the oligonucleotide and especially on the intermediate CpG. We demonstrate that the CpG step displayed 31P NMR properties similar to those found in mismatched nucleotides exclusively in the d(CTTCGAAG) duplex.