Constant pressure and temperature molecular dynamics simulation of a fully hydrated liquid crystal phase dipalmitoylphosphatidylcholine bilayer.

We report a constant pressure and temperature molecular dynamics simulation of a fully hydrated liquid crystal (L alpha) phase bilayer of dipalmitoylphosphatidylcholine at 50 degrees C and 28 water molecules/lipid. We have shown that the bilayer is stable throughout the 1550-ps simulation and have demonstrated convergence of the system dimensions. Several important aspects of the bilayer structure have been investigated and compared favorably with experimental results. For example, the average positions of specific carbon atoms along the bilayer normal agree well with neutron diffraction data, and the electron density profile is in accord with x-ray diffraction results. The hydrocarbon chain deuterium order parameters agree reasonably well with NMR results for the middles of the chains, but the simulation predicts too much order at the chain ends. In spite of the deviations in the order parameters, the hydrocarbon chain packing density appears to be essentially correct, inasmuch as the area/lipid and bilayer thickness are in agreement with the most refined experimental estimates. The deuterium order parameters for the glycerol and choline groups, as well as the phosphorus chemical shift anisotropy, are in qualitative agreement with those extracted from NMR measurements.     

Predicting the structure of the light-harvesting complex II of Rhodospirillum molischianum.

We attempted to predict through computer modeling the structure of the light-harvesting complex II (LH-II) of Rhodospirillum molischianum, before the impending publication of the structure of a homologous protein solved by means of X-ray diffraction. The protein studied is an integral membrane protein of 16 independent polypeptides, 8 alpha-apoproteins and 8 beta-apoproteins, which aggregate and bind to 24 bacteriochlorophyll-a's and 12 lycopenes. Available diffraction data of a crystal of the protein, which could not be phased due to a lack of heavy metal derivatives, served to test the predicted structure, guiding the search. In order to determine the secondary structure, hydropathy analysis was performed to identify the putative transmembrane segments and multiple sequence alignment propensity analyses were used to pinpoint the exact sites of the 20-residue-long transmembrane segment and the 4-residue-long terminal sequence at both ends, which were independently verified and improved by homology modeling. A consensus assignment for the secondary structure was derived from a combination of all the prediction methods used. Three-dimensional structures for the alpha- and the beta-apoprotein were built by comparative modeling. The resulting tertiary structures are combined, using X-PLOR, into an alpha beta dimer pair with bacteriochlorophyll-a's attached under constraints provided by site-directed mutagenesis and spectral data. The alpha beta dimer pairs were then aggregated into a quaternary structure through further molecular dynamics simulations and energy minimization. The structure of LH-II so determined is an octamer of alpha beta heterodimers forming a ring with a diameter of 70 A.     

Phosphatidylcholine and choline homeostasis

Phosphatidylcholine (PC) is made in mammalian cells from choline via the CDP-choline pathway. Animals obtain choline primarily from the diet or from the conversion of phosphatidylethanolamine (PE) to PC followed by catabolism to choline. The main fate of choline is the synthesis of PC. In addition, choline is oxidized to betaine in kidney and liver and converted to acetylcholine in the nervous system. Mice that lack choline kinase (CK) alpha die during embryogenesis, whereas mice that lack CKbeta unexpectedly develop muscular dystrophy. Mice that lack CTP:phosphocholine cytidylyltransferase (CT) alpha also die during early embryogenesis, whereas mice that lack CTbeta exhibit gonadal dysfunction. The cytidylyltransferase beta isoform also plays a role in the branching of axons of neurons. An alternative PC biosynthetic pathway in the liver uses phosphatidylethanolamine N-methyltransferase to catalyze the formation of PC from PE. Mice that lack the methyltransferase survive but die from steatohepatitis and liver failure when placed on a choline-deficient diet. Hence, choline is an essential nutrient. PC biosynthesis is required for normal very low density lipoprotein secretion from hepatocytes. Recent studies indicate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when choline supply is attenuated.     

Effect of bulky lesions on DNA: solution structure of a DNA duplex containing a cholesterol adduct

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.     

Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways

High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [³H]epibatidine ([³H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.     

Stability and amino acid preferences of type VIII reverse turn: the most common turn in peptides?

Free energies of the alpha(r)beta and betabeta conformations of 14 tetrapeptides, based on the sequence SALN and protein X-ray structures, were calculated using molecular dynamics simulations and MM-PBSA calculations. The alphaalpha conformations of five of the tetrapeptides were also studied. SALN has been earlier shown by molecular dynamics simulations and NMR spectroscopy to have a tendency to form an alpha(r)beta turn. The gas-phase energy of the molecular mechanical force field (CHARMM), the electrostatic and non-polar solvation free energies and solute entropies were used to explain the free energy differences of the alphaalpha, betabeta and alpha(r)beta conformations of the peptides. The alpha(r)beta conformation of SALN and SATN was predicted to be slightly more stable than the extended conformation (betabeta), in agreement with experimental results. The SALN mutants SAIN, SAVN, SATN, SSIN and MSHV, were also predicted to be potential alpha(r)beta turn-forming peptides. We report also revised positional potentials for the type VIII turn, based on a non-homologous set of protein structures. This protein databank analysis confirms the main results of the earlier analyses and reveals several new amino acid residues with a significant positional preference. The results of this work led us to suggest that the alpha(r)beta turn may be the most common turn type in peptides. Such turns may be readily formed in aqueous solution and thereby play important roles in the protein folding process by serving as an initiation point for structure formation.     

Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp-1

Pneumococcal bacteriophage-encoded lysins are modular choline binding proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) against streptococcal infections. Here we present the crystal structures of the free and choline bound states of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1. While the catalytic module displays an irregular (beta/alpha)(5)beta(3) barrel, the cell wall-anchoring module is formed by six similar choline binding repeats (ChBrs), arranged into two different structural regions: a left-handed superhelical domain configuring two choline binding sites, and a beta sheet domain that contributes in bringing together the whole structure. Crystallographic and site-directed mutagenesis studies allow us to propose a general catalytic mechanism for the whole glycoside hydrolase family 25. Our work provides the first complete structure of a member of the large family of choline binding proteins and reveals that ChBrs are versatile elements able to tune the evolution and specificity of the pneumococcal surface proteins.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Evolution of nicotinic acetylcholine receptor subunits

A phylogenetic tree of a gene family of nicotinic acetylcholine receptor subunits was constructed using 84 nucleotide sequences of receptor subunits from 18 different species in order to elucidate the evolutionary origin of receptor subunits. The tree constructed showed that the common ancestor of all subunits may have appeared first in the nervous system. Moreover, we suggest that the alpha 1 subunits in the muscle system originated from the common ancestor of alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, and beta 3 in the nervous system, whereas the beta 1, gamma, delta, and epsilon subunits in the muscle system shared a common ancestor with the beta 2 and beta 4 subunits in the nervous system. Using the ratio (f) of the number of nonsynonymous substitutions to that of synonymous substitutions, we predicted the functional importance of subunits. We found that the alpha 1 and alpha 7 subunits had the lowest f values in the muscle and nervous systems, respectively, indicating that very strong functional constraints work on these subunits. This is consistent with the fact that the alpha 1 subunit has sites binding to the ligand, and the alpha 7-containing receptor regulates the release of the transmitter. Moreover, the window analysis of the f values showed that strong functional constraints work on the so-called M2 region in all five types of muscle subunits. Thus, the window analysis of the f values is useful for evaluating the degree of functional constraints in not only the entire gene region, but also the within-gene subregion.      

Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells

ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).     

Unique assignment of inter-subunit association in GABA(A) alpha 1 beta 3 gamma 2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA(A) receptor (GABA(A)R alpha 1 beta 3 gamma 2). There is strong evidence that the heteropentameric receptor contains two alpha 1, two beta 3, and one gamma 2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 configurations. Here we use molecular modeling to thread the relevant GABA(A)R subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA(A) sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA(A) sequences were threaded onto the AChBP template in the gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangements. Only the gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangement satisfied three known criteria: (1) alpha 1 His(102) binds at the gamma 2 subunit interface in proximity to gamma 2 residues Thr(142), Phe(77), and Met(130); (2) alpha 1 residues 80-100 bind near gamma 2 residues 91-104; and (3) alpha 1 residues 58-67 bind near the beta 3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

Application of restrained minimization, simulated annealing and molecular dynamics simulations for the conformational analysis of oligosaccharides.

The purpose of the present study was to determine the confidence with which the small number of 1H NMR nuclear Overhauser effect (NOE) distance constraints measurable across glycosidic linkages in oligosaccharides could be used for solution conformational analysis. This was assessed by use of these constraints in restrained molecular mechanical minimization of the tetrasaccharide Gal beta 1----4(Fuc alpha 1----3)Glc-NAc beta 1----3Gal, a model compound of the Lewis-X antigenic determinant. This presents a particularly severe test case in view of extreme resonance overlap and a dearth of inter-residue distance constraints. It is concluded that these constraints, when used in conventional restrained minimization, result in the generation of 'virtual conformations' and local minima about glycosidic linkages. However, these restraints are nevertheless found to be useful in the initial stages of a conformational analysis strategy involving restrained minimization combined with dynamical simulated annealing to define more accurately the global minimum energy configuration, together with molecular dynamics simulation to explore conformational mobility about this minimum. Theoretical ROE values calculated over the time course of the MD simulation, using a formalism appropriate for the time scale of the internal motion, are compared with those obtained experimentally in the oligosaccharide.     

Three-dimensional structure of soybean beta-amylase determined at 3.0 A resolution: preliminary chain tracing of the complex with alpha-cyclodextrin.

The three-dimensional structure of a complex of soybean beta-amylase [EC 3.2.1.2] with an inhibitor, alpha-cyclodextrin, has been determined at 3.0 A resolution by X-ray diffraction analysis. Preliminary chain tracing showed that the enzyme folded into large and small domains. The large domain has a (beta alpha)8 super-secondary structure, while the smaller one is formed from two long loops extending from the beta 3 and beta 4 strands of the (beta alpha)8 structure. The interface of the two domains together with shorter loops from the (beta alpha)8 structure form a deep cleft, in which alpha-cyclodextrin binds slightly away from the center. Two maltose molecules also bind in the cleft. One shares a binding site with alpha-cyclodextrin and the other is situated more deeply in the cleft.     

Analgesic action of nicotine on tibial nerve transection (TNT)-induced mechanical allodynia through enhancement of the glycinergic inhibitory system in spinal cord

The activation of cholinergic pathways by nicotine elicits various physiological and pharmacological effects in mammals. For example, the stimulation of nicotinic acetylcholine receptors (nAChRs) leads to an antinociceptive effect. However, it remains to be elucidated which subtypes of nAChR are involved in the antinociceptive effect of nicotine on nerve injury-induced allodynia and the underlying cascades of the nAChR-mediated antiallodynic effect. In this study, we attempted to characterize the actions of nicotine at the spinal level against mechanical allodynia in an animal model of neuropathic pain, tibial nerve transection (TNT) in rats. It was found that the intrathecal injection of nicotine, RJR-2403, a selective alpha4beta2 nAChR agonist, and choline, a selective alpha7 nAChR agonist, produced an antinociceptive effect on the TNT-induced allodynia. The actions of nicotine were almost completely suppressed by pretreatment with mecamylamine, a non-selective nicotinic antagonist, or dihydro-beta-erythroidine, a selective alpha4beta2 nAChR antagonist, and partially reversed by pretreatment with methyllycaconitine, a selective alpha7 nAChR antagonist. Furthermore, pretreatment with strychnine, a glycine receptor antagonist, blocked the antinociception induced by nicotine, RJR-2403, and choline. On the other hand, the GABAA antagonist bicuculline did not reverse the antiallodynic effect of nicotine. Together, these results indicate that the alpha4beta2 and alpha7 nAChR system, by enhancing the activities of glycinergic neurons at the spinal level, exerts a suppressive effect on the nociceptive transduction in neuropathic pain.     

Cytokine Regulation of Nerve Growth Factor-Mediated Cholinergic Neurotrophic Activity Synthesized by Astrocytes and Fibroblasts

The neurotrophic activity of astrocytes and fibroblasts and its regulation by various cytokines were investigated. Astrocyte conditioned medium (ACM) enhanced the survival of neurons and the proliferation of astrocytes in embryonic cortical cultures grown in serum-free defined medium. However, these results were not affected by acidic fibroblast growth factor, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), and transforming growth factor-beta 1. In contrast, ACM induced choline acetyltransferase expression in septal cholinergic neurons via nerve growth factor (NGF)-dependent and -independent mechanisms. However, neither acidic nor basic fibroblast growth factor is involved in this biological activity in ACM. The cytokines listed above mainly stimulate NGF-mediated cholinergic neurotrophic activity in ACM. A combination of IL-1 beta and TNF alpha significantly enhanced choline acetyltransferase activity in septal neurons co-cultured with astrocytes, and this effect was found to be mediated by NGF produced by activated astrocytes. Effects of astrocytes on GABAergic neurons were also examined. ACM was found to increase glutamate decarboxylase activity in neuronal cultures from septum in the presence of Ara-C. However, the cytokines did not enhance this activity in ACM. Moreover, a combination of IL-1 beta and TNF alpha had no effect on glutamate decarboxylase activity in septal neurons co-cultured with astrocytes. In a final set of experiments, cholinergic neurotrophic activity in skin-derived fibroblast conditioned medium (FCM) was examined. FCM was found to possess biological activity similar to that of ACM on septal neurons grown in serum-free defined medium with Ara-C. The cytokines also enhanced NGF-mediated cholinergic neurotrophic activity in FCM. Astrocytes and fibroblasts were found to possess NGF-type and non-NGF-type cholinergic neurotrophic activity, and various cytokines were found to regulate the NGF-type cholinergic neurotrophic activity in both types of cells. NGF produced by astrocytes and fibroblasts that are activated by cytokines is likely to be important for development and regeneration of NGF-sensitive neurons in the central and peripheral nervous systems.     

A dominant negative mutant beta 2-microglobulin blocks the extracellular folding of a major histocompatibility complex class I heavy chain

The major histocompatibility complex class I (MHC1) molecule plays a crucial role in cytotoxic lymphocyte function. beta 2-Microglobulin (beta 2m) has been demonstrated to be both a structural component of the MHC1 complex and a chaperone-like molecule for MHC1 folding. beta 2m binding to an isolated alpha 3 domain of MHC1 heavy chain at micromolar concentrations has been shown to accurately model the biochemistry and thermodynamics of beta 2m-driven MHC1 folding. These results suggested a model in which the chaperone-like role of beta 2m is dependent on initial binding to the alpha 3 domain interface of MHC1 with beta 2m. Such a model predicts that a mutant beta 2m molecule with an intact MHC1 alpha 3 domain interaction but a defective MHC1 alpha 1 alpha 2 domain interaction would block beta2m-driven folding of MHC1. In this study we generated such a beta 2m mutant and demonstrated that it blocks MHC1 folding by normal beta 2m at the expected micromolar concentrations. Our data support an initial interaction of beta 2m with the MHC1 alpha 3 domain in MHC1 folding. In addition, the dominant negative mutant beta 2m can block T-cell functional responses to antigenic peptide and MHC1.     

Hydrostatic pressure-induced conformational changes in phosphatidylcholine headgroups: a 2H NMR study.

The effects of pressure and temperature on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine headgroup conformations were examined using deuterium nuclear magnetic resonance. Isothermal compression was found to produce a decrease in the choline alpha deuteron quadrupole splitting and increases in the choline beta and gamma deuteron quadrupole splittings. A similar counterdirectional change, seen in the presence of positive surface charge, has been attributed to tilting of the headgroup away from the bilayer surface in response to the torque exerted on the phosphocholine dipole by positive surface charges. The direction of the change in headgroup deuteron quadrupole splitting is consistent with the pressure-induced reduction in area per lipid in the liquid crystalline phase, which can be inferred from the ordering of phospholipid acyl chains under comparable conditions. The temperature dependences of the headgroup deuteron quadrupole splittings were also examined. It was found that at elevated pressure, the alpha splitting was insensitive to temperature, whereas the beta and gamma splittings decreased. The response of the beta deuteron splitting to temperature was found to be weaker at elevated pressure than at ambient pressure.     

Tertiary structure in N-linked oligosaccharides

Distance constraints derived from two-dimensional nuclear Overhauser effect measurements have been used to define the orientation of the Man alpha 1-3Man beta linkage in seven different N-linked oligosaccharides, all containing the common pentasaccharide core Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. Conformational invariance of the Man alpha 1-3Man beta linkage was found for those structures bearing substitutions on the Man alpha 1-3Man beta antenna. However, the presence of either a GlcNAc residue in the beta 1-4 linkage to Man beta ("bisecting GlcNAc") or a xylose residue in the beta 1-2 linkage to Man beta of the trimannosyl core was found to generate conformational transitions that were similar. These transitions were accompanied by characteristic chemical shift perturbations of proton resonances in the vicinity of the Man alpha 1-3Man beta linkage. Molecular orbital energy calculations suggest that the conformational transition between the unsubstituted and substituted cores arises from energetic constraints in the vicinity of the Man alpha 1-3Man beta linkage, rather than specific long-range interactions. These data taken together with our previous results on the Man alpha 1-6Man beta linkage [Homans, S. W., Dwek R. A., Boyd, J., Mahmoudian, M., Richards, W. G., & Rademacher, T. W. (1986) Biochemistry 25, 6342] allow us to discuss the consequences of the modulation of oligosaccharide solution conformations.     

Solution conformations of GM3 gangliosides containing different sialic acid residues as revealed by NOE-based distance mapping, molecular mechanics, and molecular dynamics calculations.

The conformation of the GM3 ganglioside, Neu5Ac alpha 2-3Gal beta 1-4Glc beta 1-1 Cer, and its analogs containing the Neu5Gc or Neu4Ac5Gc residues (Gc = glycolyl, CH2OHCO) was investigated in Me2SO-d6 solution with the aid of a distance-mapping procedure based on rotating-frame NOE contacts, with hydroxyl protons being used as long-range sensors defining the distance constraints. A pronounced flexibility found for both the Neu-Gal and Gal-Glc linkages was confirmed by 1000-ps molecular dynamics simulations. Similar results, although based on a smaller number of NOE constraints, were obtained for GM3 gangliosides anchored in mixed D2O/dodecylphosphocholine-d38 micelles and for the Neu5Ac-, Neu5Gc-, and Neu5,9Ac2-sialyllactoses dissolved in D2O. No noteworthy differences in conformational behavior of the glycan chains of the three gangliosides or sialyllactoses were observed in either of the media.