Conformational analysis of inulobiose by molecular mechanics

Conformational energies for inulobiose [beta-D-fructofuranosyl-(2----1)-beta-D-fructofuranoside], a model for inulin, were computed with the molecular mechanics program MMP2(85). The torsion angles of the three linkage bonds were driven in 20 degree increments, and the steric energy of all other parameters was minimized. The linkage torsion angles defined by C-1'-C-2'-O-C-1 (phi) and O-C-1-C-2-O-2 (omega) have minima at +60 degrees and -60 degrees, respectively, regardless of side group orientation; accessible minima exist at other staggered conformations. The torsion angle at the central bond C-2'-O-1-C-1-C-2 (psi) was approximately 180 degrees in all the low-energy conformers. This appears to be generally true for rings linked by three bonds. The fructofuranose rings initially had low-energy 4/3T conformations (angle of pseudorotation, phi 2 = 265 degrees) that were retained except when the linkage conformations created severe inter-residue conflicts. In those cases, almost all puckerings of the furanose rings were found.     

Conformational analysis of levanbiose by molecular mechanics.

A relaxed conformational energy map for levanbiose, O-beta-D-fructofuranosyl-(2----6)-beta-D-fructofuranoside, was computed with the molecular mechanics program MM2(87). All torsion angles of the three linkage bonds were driven by 30 degrees increments while two primary alcohol groups were held at three staggered forms. The steric energy of all other parameters was optimized. The side groups were retained at the same relative positions on the two rings in this first part of the study so our results are directly applicable to the study of polymeric levan with identical repeating units. The low-energy dimers did not lead to viable polymers. The interresidue linkage torsion angles defined by C-6-O-2'-C-2'-C-1' (phi) and O-5-C-5-C-6-O-2' (omega) have minima at +60 degrees and -60 degrees, respectively, with accessible minima at other staggered forms. As observed in inulobiose, the preferred torsion angle at central linkage bond defined by C-5-C-6-O-2'-C-2' (psi) was antiperiplanar. An analysis of all conformations of staggered side groups showed that the C-1 and C-1' groups had little effect but the C-6' group showed a preference for chi-6'(O-5'-C-5'-C-6'-O-6') = -60 degrees. The fructofuranose rings were started at the low-energy 4(3)T conformation (angle of pseudorotation, phi 2 = 265 degrees) that was retained except when the linkage conformations created severe inter-residue conflict.     

Recognition of ribonuclease A by 3'-5'-pyrophosphate-linked dinucleotide inhibitors: a molecular dynamics/continuum electrostatics analysis

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5′-phospho-2′-deoxyuridine-3-pyrophosphate (P→5)-adenosine-3-phosphate (pdUppA-3′-p). The 3′,5′-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B₂ and the placement of the 5′-β-phosphate of the adenylate (instead of the α-phosphate) at subsite P₁ where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3′-p and the related weaker inhibitor dUppA, which lacks the 3′ and 5′ terminal phosphate groups of pdUppA-3′-p. The simulations show that the adenylate 5′-β-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P₁ and B₂. The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3′-p varies between −7.9 kcal/mol and −2.8 kcal/mol for a range of protein/ligand dielectric constants (ε_p) 2–20, in good agreement with the experimental value (−3.6 kcal/mol); the agreement becomes exact with ε_p = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3′-p make increased interactions with residues Lys⁷ and Lys⁶⁶ of the more remote sites P₂ and P₀, and His¹¹⁹ of site P₁.     

Conformational analysis of the trinucleoside diphosphate 3'd(A2'-5'A2'-5'A). An NMR and CD study.

A 500 MHz and 300 MHz NMR study of the trinucleoside diphosphate 3'd(A2'-5'A2'-5'A) is presented. In addition, circular dichroism is used to study base stacking in the title compound. The complete 1H-NMR spectral assignment of the sugar ring proton signals is given. Information about the sugar ring (N- or S-type conformation) and about the backbone geometry along C4'-C5' and C5'-O5' bonds is obtained from the NMR coupling constants. It is shown that the trimer mainly occurs in the N-N-N stacked state at low temperatures; the presence of a minor amount of N-N-S conformational sequence is indicated.     

Synthesis of the E. coli pyruvate dehydrogenase complex: non-dependence on 3'-5'-cyclic AMP

A determination of the level of the pyruvate dehydrogenase complex in a 3′–5′-c-AMP deficient mutant of $\text{E.}$$\text{coli}$ K12 has been carried out. The deficiency has no effect on specific activities for derivatives carrying either the inducible genes for two components of the complex or constitutive mutants. We conclude that synthesis of the complex is not sensitive to catabolite repression.     

NMR solution structures of [d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2] and its unmodified control.

We present the high-resolution solution structures of a self-complementary DNA decamer duplex featuring a single alpha-anomeric nucleotide per strand encompassed by a set of 3'-3' and 5'-5' phosphodiester linkages, d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2, alphaT, and its unmodified control, d(GCGAATTCGC)2, obtained by restrained molecular dynamics. Interproton distance and deoxyribose ring torsion angle restraints were deduced from homonuclear NOESY and DQF-COSY data, respectively. For both the control and alphaT duplexes, excellent global convergence was observed from two different (A- and B-) starting models. The final average structures of the two duplexes are highly homologous, and overall possess the traits characteristic of right-handed B-DNA duplexes. However, localized differences between the two structures stem from the enhanced conformational exchange in the deoxyribose ring of the cytidine following the 5'-5' linkage, the C3'- exo pseudorotation phase angle of the alpha-nucleotide, and unusual backbone torsions in the 3'-3' and 5'-5' phosphodiester linkages. The structural data reported here are relevant to the design of antisense therapeutics comprised of these modifications.     

The effect of pyridoxal-5-phosphate on the inhibition of platelet aggregation and adenosine-3'-5'-cyclic monophosphate accumulation in human platelets

Platelet adenosine-3'-5'-cyclic monophosphate (c-AMP) was determined in platelet rich plasma and washed platelets by a modification of the c-AMP protein binding assay. Studies were performed in the presence and absence of platelet aggregation inducers (adenosine diphosphate and thrombin) and inhibitors (pyridoxal-5-phosphate (PALP), prostaglandin E1 (PGE1), theophylline and papaverine). At 70% inhibitory concentration of platelet aggregation (PA) induced by papaverine or theophylline, a small but significant increase in platelet c-AMP level was found. With PALP however the inhibition of PA was not associated with a significant increase in the c-AMP level. PALP or theophylline potentiated greatly both the inhibition of PA and c-AMP accumulation induced by PGE1. Yet PALP potentiated the inhibition of PA induced by theophylline without increasing platelet c-AMP level. Our results indicate that the inhibition of PA by PALP is not mediated by c-AMP accumulation. However in the presence of PGE1, the increment in PA inhibition, is mediated by further indirect activation of the adenyl-cyclase.     

Content of adenosine 3'-5'-cyclic monophosphate in the pancreatic islets of mice with a hereditary defect of insulin secretion

Glucose (20 mM) released insulin from pancreatic islets of $\text{C57BL}\text{6J}\text{-}\text{db}{}^{\mathrm{2J}}\text{db}{}^{\mathrm{2J}}$ mice, the response being potentiated by 1 mM 3-isobutyl-1-methylxanthine. Islets of $\text{C57BL}\text{KsJ}\text{-}\text{db}\text{db}$ mice failed to respond to glucose and released only little insulin when challenged with both glucose and methylxanthine. After incubation with 0 or 20 mM glucose alone the islet content of adenosine 3′:5′-cyclic monophosphate did not differ between the two types of mice or between glucose concentrations. 3-Isobutyl-1-methylxanthine increased the islet adenosine 3′:5′-cyclic monophosphate markedly in $\text{6J-}\text{db}{}^{\mathrm{2J}}\text{db}{}^{\mathrm{2J}}$ mice but not significantly in $\text{KsJ-}\text{db}\text{db}$ mice.     

Prostacyclin-induced activation of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase in rat antral and fundic gastric mucosa.

The present investigations showed that after oral prostacyclin administration (100 micrograms/kg) as soon as the intracellular level of cAMP is elevated the activation of cAMP-dependent protein kinase follows in both parts (antrum and fundus) of rat gastric mucosa. The enzyme activation seems to be more significant in the fundic region which is in a complete agreement with the previously published results, i.e. the fundic mucosa reacts with de novo protein synthesis toward noxious agents (resulting finally in new cell formation), while the antral mucosa is more durable against damaging noxae. Taking into consideration all available data in the literature it seems that the intracellular effect of the exogenously administered prostacyclin in the gastric mucosa is a polyphasic effect, which contains the following consecutive steps: 1. Binding to the cell surface; 2. Effect on the intracellular second messenger system, (cAMP, cGMP); 3. Activation of the calmodulin system; 4. cAMP-dependent protein kinase activation; 5. DNA, RNA changes; 6. Influence on protein synthesis, and finally; 7. New cell formation.     

Conformational analysis of endomorphin-2 by molecular dynamics methods

Endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH(2)) is a highly potent and selective mu-opioid receptor agonist. A conformational analysis of EM2 was carried out by simulated annealing (SA) and molecular dynamics (MD) methods. Molecular modeling was conducted on both neutral (N-terminal NH(2)) and charged (N-terminal NH(3) (+)) molecules. Based on the results of NMR investigations showing an equilibrium mixture of cis and trans Tyr(1)-Pro(2) peptide bonds for EM2 in solution, simulations were performed with restrained cis-Pro and trans-Pro peptide bonds, too. A separate SA study with unrestrained Pro peptide bonds was also conducted. Preferred conformational states are presented in Ramachandran plots. The g(+), g(-), and trans populations of the aromatic amino acid residue side chains were determined in chi(1) space. The distances between the N-terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic rings and the Pro(2) ring, were determined. The energy distribution of the structures obtained by SA was calculated. The preferred secondary structural elements were different kinds of beta-turns, an inverse gamma-turn located in the N-terminal region, and regular and inverse gamma-turns located in the C-terminal region. These turns were stabilized by intramolecular H-bonds and bifurcated H-bonds.     

Conformational studies of some 2':3'-cyclic mononucleotides in solution by different nuclear-magnetic-resonance methods

The conformations of the 2':3'-cyclic mononucleotides of adenosine and cytidine in deuterium oxide has been studied at pH 2.3, using lanthanide ions as paramagnetic nuclear magnetic resonance (NMR) probes. It was not possible to find any single conformation for these molecules which accounts for the observed shift and relaxation data. This situation is in agreement with the interpretation of vicinal 1H-1H and 1H-31P coupling constants, which indicate that the ribofuranose and cyclic phosphate rings are in rapid equilibrium between different puckered forms. The interpretation of the lanthanide data in terms of an equilibrium between different conformations give average rotamer populations in good agreement with the coupling constant analysis. The conformations of these systems in aqueous solutions were found to be more flexible than in the solid state, where rigid planar ribofuranose rings have been observed. Adenosine 2':3'-monophosphate differs from cytidine 2':3'-monophosphate at the glycosidic link.     

Conformational analysis of a dinucleotide photodimer with the aid of the genetic algorithm.

The solution structure of the photodimer cis,syn-dUp[]dT is derived with the aid of the genetic algorithm. The conformational space available for the molecule is sampled efficiently using the computer program DENISE and tested against a set of constraints available from nmr experiments. The dominant conformation in solution found with this approach can be described by the following combinations of sugar-phosphate backbone torsion angles: epsilon(t), zeta(t), alpha(+), beta(-ac), and gamma(t). The conformation of the sugars and glycosidic torsion angles are S type and syn, respectively. The cyclobutane ring and pyrimidines are puckered. In addition, other conformations that exist in equilibrium with the first are found. It is concluded that the cyclobutane-pyrimidine system is rigid, whereas the sugar-phosphate backbone is flexible. The solution structures are compared with the crystal structure of the strongly related cyano-ethyl ester of cis,syn-dTp[]dT.     

Conformational characteristics of the trinucleoside diphosphate xyloA2'-5'xyloA2'-5'xyloA. A nuclear magnetic resonance and CD study.

In this paper the conformational analysis of the 2'-5' linked xylotrinucleotide xA2'-5'xA2'-5'xA is reported. The title compound is an analogue of A2'-5'A2'-5'A, which compound was shown to display inhibitive effects on protein synthesis. The complete 1H-NMR assignment of the high field spectral region of the xylose trimer is given. Modes of base-base stacking are extracted from coupling constant data at various temperatures. Circular dichroic (CD) spectra confirm the presence of stacked states at low temperature. Xylonucleosides are known to prefer the N-type sugar conformation. However, in the present trimer the S-type conformer is suggested to partake in stacked conformations. Two types of stacking in the two constituent dimer fragments of the trimer are proposed to rationalize the NMR data: xA(1)N-xA(2)S and xA(2)N-xA(3)S.     

Conformational study of the pentadecapeptide 2F10 by circular dichroism, IR spectroscopy and molecular mechanics

The conformational profile of the pentadecapeptide of sequenceAVYYCTRGYHGSSLY, capable of mimicking the group-specific a determinant of human hepatitis B surface antigen at both the B- and T-cell level, was assessed using the combined informationprovided by circular dichroism (CD) studies, IR spectroscopy and molecular mechanics. Specifically, the CD spectra of the peptide was recorded in various environments including an aqueous buffer, trifluoroethanol, hexafluoroisopropanol and inmicellar solutions of sodium dodecylsulfate in order to analyzeits conformational profile. Analysis of the results suggests the strong tendency of the peptide to adopt structures in the different structuring media. Furthermore, the IR spectrumof the peptide recorded in DMSO shows absorptions compatible with a sheet structure. Finally, molecular mechanics calculations using an iterative simulated annealing protocol to sample the conformational space, supplemented by a moleculardynamics simulation in water, suggest as the most important peptide secondary structure feature the adoption of a hairpinconformation. Accordingly, the combined information provided by the different techniques used in the present work, consistently suggest that the peptide 2F10 exhibits a tendencyto adopt a hairpin conformation in solution.