Conformational study of the acetyl group and cyclohexenone in progesterone interacting with phosphatidyllipid by means of circular dichroism

The interaction between the A-ring and the 17-acetyl groups of progesterone (PROG) and various concentrations of distearoyl-, dipalmitoyl-, dioleoyl- and diarachidoyl-L-alpha-phosphatidylcholines, and dipalmitoyl-L-alpha-phosphatidyl-DL glycerol in methanol and chloroform solutions and its preferred conformational assignments in the presence of those lipids were examined qualitatively by circular dichroism on the basis of PROG spectra in the wavelength regions of 260-400 nm. PROG did not interact with saturated distearoyl and dipalmitoyl phosphatidylcholines, but did with unsaturated dioleoyl and diarachidoyl phosphatidylcholines, and saturated dipalmitoylphosphatidylglycerol. The interacting moieties of PROG were an alpha,beta-unsaturated cyclohexenone of the A-ring for oleoyl and glycerol lipids, and the 17-acetyl group for unsaturated and glycerol lipids. The interaction with these lipids, the rotational conformations of the 17-acetyl group, and invertible conformations of the cyclohexenone of PROG were discussed on the basis of the elliptical strength of the Cotton effect and energy estimation of the preferred conformers. Oleoylphosphatidylcholine caused an increase in slightly energetically unstable conformers of the acetyl group and stable conformers of the alpha,beta-unsaturated cyclohexenone. Glycerol lipid, on the other hand, caused an increase in energetically unstable conformers of cyclohexenone, but it was similar to the effect of oleoyl lipid on the 17-acetyl group. Diarachidoyl-L-alpha-phosphatidylcholine, with eight double bonds, other hand, increased the number of energetically stable conformers of the 17-acetyl group, but had no effect on the conformation of cyclohexenone. It became apparent that the double bond of hydrocarbon moiety as well as the head group of choline and glycerol in lipids were closely related to the conformational populations of both groups of the PROG molecule. The specific effect on the conformations of the acetyl and alpha,beta-unsaturated cyclohexenone of PROG of various lipids with different substitutions in their heads or hydrocarbon moieties might in part explain the nongenomic action of the steroid.     

The X-ray investigation of 16 alpha,17 alpha-thiazolidine derivatives of delta 4- and delta 5-pregnanes and conformational analysis of their oxathiolane analog

An X-ray study of 3,20-dioxo-4-pregnene-[16 alpha,17 alpha-d]--2',2'-dimethylthiazolidine (I) and 3 beta-hydroxy-20-oxo-5--pregnene-[16 alpha,17 alpha-d]-2',2'- dimethylthiazolidine (II) has been carried out. Two independent molecules in crystal II have significantly different conformations of the D and E rings, although according to the atom-atom potential calculations the energy of interaction of these molecules with their neighbors in crystal is the same. The calculation of conformational energy of 3,20-dioxo-4-pregnene-[17 alpha,16 alpha-d]-2',2'--dimethyloxathiolane (III) by the molecular mechanics method (MMM) indicates a possibility of existence of two similar conformers also for this molecule. The MMM calculation shows also that the conformation of molecule III (as well as progesterone) with the 17 beta-acetyl group torsion angle C(16)C(17)C(20)0(20) close to -120 degrees is possible.     

Synthesis and conformational analysis of cyclo-TRI[l-valyl-d-hexahydromandelyl]

The synthesis of the cyclo-hexadepsipeptide [l-valyl-d-hexahydromandelyl]₃ is described. Examination of this macrocyclic compound by 220-MHz nuclear magnetic resonance spectroscopy shows that symmetrical conformations are stabilized in strongly polar solvents (trifluoroacetic acid, acetonitrile), whereas asymmetric conformations are preferred in nonpolar or slightly polar media such as carbon tetrachloride, chloroform, cyclohexane, and benzene.From analysis of the temperature dependence of the chemical shift and of the coupling constants, together with conformational energy calculations, a model is proposed for the preferred conformation of this molecule in nonpolar solvents.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-6)-linked moiety

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of the alpha(1-6)-linked moiety is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions and to draw conclusions about the behavior in solution of these molecules. For all classes, identical conformations were found for the 6-arm except for the torsional angle, omega, about the C5-C6 bond of the alpha 1-6 linkage. For high mannose and hybrid structures omega was found to be -60 degrees, for bisected biantennary complex structures omega was 180 degrees, and for complex biantennary structures averaging between -60 degrees and 180 degrees occurs.     

Comparative conformational analysis of cholesterol and ergosterol by molecular mechanics

A comparative conformational analysis of cholesterol and ergosterol has been carried out using molecular mechanics methods. These studies are aimed at giving a better understanding of the molecular nature of the interaction of these sterols with polyene macrolide antibiotics. Structures of cholesterol and ergosterol determined by X-ray methods have been used as initial geometries of these molecules for force field calculations. The calculation of steric energy has also been made for conformations which do not appear in the crystal. The latter conformers have different conformations of the side chain as well as different conformations of rings A and D. The rotational barriers around bonds C17–C20 and C20–C22 have also been calculated. The results obtained on differences and similarities in the conformations of cholesterol and ergosterol allow us to postulate a mechanism for differential interaction with the antibiotics. The relatively rigid side chain of ergosterol (stretched molecule) in comparison with the flexible side chain of cholesterol (bent molecule), allows better intermolecular contact of the first sterol molecule with a polyene macrolide and in consequence facilitates complex formation involving Van der Waal's forces.     

NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of human [113Cd7]metallothionein-2 was determined by nuclear magnetic resonance spectroscopy in solution. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling constants 3JHN alpha and 3J alpha beta. The molecule consists of two domains, the beta-domain including amino acid residues 1 to 30 and three metal ions, and the alpha-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.     

Solution conformation of sialosylcerebroside (GM4) and its NeuAc(alpha 2----3)Gal beta sugar component

The solution conformations of GM4 ganglioside [NeuAc(alpha 2----3)Gal (beta 1----1)Cer] in (2H3C)2SO and its component disaccharide in 2H2O were investigated with the aid of 1H-nuclear magnetic resonance spectroscopy (nuclear Overhauser effect, analysis of coupling constants) and by energy-minimum calculations. The existence of three low-energy conformers obtained by theoretical calculations was supported by experimental findings in the case of GM4, whereas the disaccharide appears to exist as a mixture of two conformers.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.     

Conformational energy calculations of enzyme-substrate complexes of lysozyme. I. Energy minimization of monosaccharide and oligosaccharide inhibitors and substrates of lysozyme

Conformational energy calculations were performed on monosaccharide and oligosaccharide inhibitors and substrates of lysozyme to examine the preferred conformations of these molecules. A grid-search method was used to locate all of the low-energy conformational regions for N-acetyl-β-D -glycosamine (NAG), and energy minimization was then carried out in each of these regions. Three stable positions for the N-acetyl group have ben located, in two of which the plane of the amide unit is normal to the mean plane of the pyranosyl ring. Nine local energy minima were located for the —CH₂OH group. The positions of the two vicinal cis —OH groups are determined predominantly by interactions with either the —CH₂OH or the N-acetyl group. The most stable conformations of β-N-acetylmuramic acid (NAM) were determined from the study of the low-energy conformations of NAG. In the two stable orientations for the D -lactic acid side chain, the O—C—C′ plane (C′ being the carbon atom of the terminal carboxyl group) was found to be normal to the mean plane of the pyranosyl ring. The low-energy positions for the COOH group of NAM are determined mainly by interactions with neighboring groups. The conformational preferences of the α-anomers of NAG and NAM were also explored. The calculated conformation of the N-acetyl group for α-NAG was quite close to that determined by X-ray analysis. Two of the three lowest energy conformations of α-NAM are similar to the corresponding conformations of the β-anomer. A third low-energy structure, which has a hydrogen bond from the NH of the N-acetyl group to the C?O of the lactic acid group, corresponds very closely to the X-ray structure of this molecule. The preferred conformations of the disaccharides NAG–NAG, NAM–NAG and NAG–NAM were also investigated. Two preferred orientations of the reducing pyranosyl ring relative to the nonreducing ring were found for all of these disaccharides, both of which are close to the extended conformation. In one of these conformations, a hydrogen bond can form between the OH group attached to C₃ of the reducing sugar and the ring oxygen of the preceding residue. Each conformation can be stabilized further by a hydrogen bond between the CH₂OH (donor) of residue i + 1 and the C?O of residue i (acceptor). The interactions that determine conformations for all oligosaccharides containing both NAG and NAM are shown to be exclusively intraresidue and nearest neighbor interactions, so that it is possible to predict all stable conformations of oligosaccharides containing NAG and NAM in any sequence.     

The detailed conformational study of a leukotriene antagonist, FPL-55712, using high-field nuclear magnetic resonance spectroscopy

High-field proton magnetic resonance measurements at 400 MHz and 600 MHz allowed the evaluation of the preferred conformations of a leukotriene antagonist, FPL-55712. The experiments involved an analysis of proton-proton coupling constants, longitudinal relaxation time data and nuclear Overhauser effect experiments. The NMR parameters confirm the conformational features expected from X-ray and microwave data for related substances, such as rotational freedom about C14-C15 and C15-C16, synperiplanar arrangements for C7-C8-O-C14 and C16-O-C17-C18 and segmental motion in the propyl side chains.     

Interactive NMR and computer simulation studies of lanthionine-ring structures

We report progress in elucidating the structure of nisin, a naturally occurring peptide antibiotic. Nisin contains five rings constrained by lanthionine or methyllanthionine bridges, as well as alpha, beta-unsaturated amino acids. We have determined conformations for two model compounds of ring A and a derivative of ring B through interactive nmr and computer simulation studies. High-resolution nmr techniques provides structural information, which was further refined through molecular dynamics simulations. These methods are being applied to the remaining constrained fragments of the molecule. This conformational information will be employed in an aufbau approach to determining the structure of the entire molecule.     

Conformational investigation of alpha,beta-dehydropeptides. Part. IV. Beta-turn in saturated and alpha,beta-unsaturated peptides Ac-Pro-Xaa-NHCH3: NMR and IR studies.

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Val, Phe, Leu, Abu, Ala) as well as alpha,beta-unsaturated Ac-Pro-delta Xaa-NHCH3 [delta Xaa = delta Val, (Z)-delta Phe, (Z)-delta Leu, (Z)-delta Abu] were investigated in CDCl3 and CH2Cl2 by 1H-, 13C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts delta delta for NH (Xaa) and NHCH3 on going from CDCl3 to (CD3)2SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and alpha,beta-dehydropeptides (delta Xaa) on the other. Former compounds are conformationally flexible with an inverse gamma-bend, a beta-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and alpha,beta-dehydropeptides are very similar, with the type-II beta-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The beta-turn formation propensity seems to be somewhat greater in alpha,beta-unsaturated than in heterochiral peptides, but an estimation of beta-folded conformers is risky.     

The Detailed Conformational Study of a Leukotriene Antagonist,FPL-55712, using High-Field Nuclear Magnetic Resonance Spectroscopy

Abstract

High-field proton magnetic resonance measurements at 400 MHz and 600 MHz allowed the evaluation of the preferred conformations of a leukotriene antagonist, FPL-55712. The experiments involved an analysis of proton-proton coupling constants, longitudinal relaxation time data and nuclear Overhauser effect experiments. The NMR parameters confirm the conformational features expected from X-ray and microwave data for related substances, such as rotational freedom about C14—C15 and C15—C16, synperiplanar arrangements for C7—C8—O—C14 and C16—O—C17—C18 and segmental motion in the propyl side chains.     

Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study

The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.     

Quantitative determination of the conformation of ATP in aqueous solution using the lanthanide cations as nuclear-magnetic-resonance probes.

Chemical shift perturbations of the eight 1H resonances and of the three 31P resonances in the nuclear magnetic resonance spectra of ATP in 2H2O, pH 6.0, have been induced by specifically bound lanthanide cations Ln3+ (Ln = Pr, Nd, Eu, Yb). After separation of contact (through bond) perturbations the resultant through-space shifts, which are found to have axial symmetry, are used in an analysis of the conformation of the Ln3+ -ATP complex. A computer program was used to search for the conformations of the molecule which fit the nuclear magnetic resonance data. The "best" solutions obtained represent a small closely interrelated family of conformations. Effects of the cation Gd3+ on the longitudinal relaxation rates of five of the protons of ATP were also measured and used to confirm the conformational family. One of these conformations corresponds closely to one of the crystal structure forms, with an anti arrangement of the base-ribose unit and and a right-hand helical phosphate chain folded towards the adenine part of the molecule. The lanthanide ion binds predominantly to the beta and gamma phosphates and does not interact with the purine ring, these two centres being separated by at least one water molecule.     

The spatial structure of the axially bound methionine in solution conformations of horse ferrocytochrome c and Pseudomonas aeruginosa ferrocytochrome c 551 by 1H NMR

A generally applicable method for the determination of the spatial structure of the heme iron-bound methionine in c-type ferrocytochromes at atomic resolution is presented. It relies primarily on measurements of nuclear Overhauser effects between the individual hydrogen atoms of the axial methionine, and between individual hydrogens of the methionine and the heme group. Four different methionine conformers, corresponding to the four possible stereospecific assignments for the methionine methylene proton resonances, are generated by a structural interpretation of the nuclear Overhauser effects with the use of an interactive computer graphics technique. A unique structure and unique stereospecific resonance assignments are then obtained by discriminating between these four conformers on the basis of van der Waals' constraints and heme ring current effects on the chemical shifts. The use of the method is illustrated with studies of horse ferrocytochrome c and Pseudomonas aeruginosa ferrocytochrome c 551. Comparison with the crystal structures shows close coincidence between the methionine conformations in solution and in single crystals of these proteins.Abbreviations NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - TOE truncated driven nuclear Overhauser effect     

Nuclear magnetic resonance analysis and conformational characterization of a cyclic decapeptide antagonist of gonadotropin-releasing hormone

Two-dimensional proton nuclear magnetic resonance spectroscopy at 500 MHz has been carried out on the cyclic decapeptide antagonist of gonadotropin-releasing hormone: cyclo-(delta 3-Pro1-D-pClPhe2-D-Trp3-Ser4-Tyr5-D-Trp6-NMeLeu7-Arg8- Pro9-beta-Ala 10). The antagonist exists in two slowly interconverting conformations. All data are consistent with the conclusion that one form has all-trans peptide bonds and the other has a cis beta-Ala10-delta3-Pro1 bond. With the use of sequential assignment methods, chemical shift assignments were obtained for all backbone and side-chain protons of both conformational isomers except for the serine and tyrosine hydroxyl groups and the C gamma, C delta, and guanidinium group protons of the arginine. Temperature dependence of spectral parameters and magnitudes of observed nuclear Overhauser effects support the interpretation that both conformers of the antagonist consist of two beta-turns (type II', D-Trp6-NMeLeu7; type II, delta 3-Pro1-D-pClPhe2) connected by extended antiparallel beta-like strands.     

Relation of the progestagenic activity and conformation of the 17beta-acetyl side chain in the D'6-pentarane series

The synthesis of 2' beta-methyl-16 alpha,17 alpha-cyclohexanoprogesterone and its MM2 conformational analysis have been performed. The acetyl side chain was shown to have an unusual conformation with the torsion angle C13-C17-C20-O20 being -32.1 degrees. This conformation is by 5.4 kJ.mol-1 more stable than the usual one with the torsion angle 130.3 degrees. 2' beta-Methyl-16 alpha,17 alpha-cyclohexanoprogesterone proved to be inactive as a progestogen (pregnancy maintenance and McPhail tests). The lack of the activity may be due to the additional methyl group in D'-ring causing a change of the conformation of the 17 beta-acetyl side chain, thus hindering the formation of the conformation necessary for binding to the progesterone receptor.     

Quantitative determination of conformations of flexible molecules in solution using lanthanide ions as nuclear magnetic resonance probes: application to adenosine-5'-monophosphate

A procedure is described here whereby the conformation, of a flexible molecule in solution can be found. The method depends on the study of the nuclear magnetic resonance spectrum of the molecule in the presence of perturbations due to specifically bound lanthanide cations. The magnetic perturbations are of two kinds: shifts of nuclear magnetic resonance spectral lines in the presence of cations such as Eu³⁺ and changes in relaxation rates of the nuclear magnetic resonance excitations in the presence of cations such as Gd³⁺. Suitable expressions are given for the relation between the magnitude of the perturbations and the geometry of the lanthanide complex in the absence of through-bond perturbations and for an axially symmetric system. It is proved that the spectral changes described here are not due to through-bond (contact) effects. The circumstances, in which the anisotropy of the magnetic susceptibility tensor, as seen in the nuclear magnetic resonance spectra, is of axial symmetry, are defined. The experimental systems described are of this kind. A computer program has been devised that searches for the conformations of the molecule which fit the nuclear magnetic resonance data.We outline here the principles of the method and how we have used a combination of relaxation and shift probes to obtain the conformation of adenosine-5′-monophosphate at pH 2. It is shown that a small family of closely related conformations fit the nuclear magnetic resonance data. These conformations are very similar to that of the crystal structure of AMP.