Adducts of antimony(V) pentachloride with neutral lewis bases. Part II. Kinetics and reaction mechanism: a variable temperature and variable pressure proton NMR study

The ligand exchange reaction SbCl₅·L + ^*L⇌ SbCl₅·^*L + L has been studied in CH₂Cl₂ and (CHCl₂)₂ as a function of temperature and of pressure by using ¹H NMR. First order rate laws, positive activation entropies ranging from +9.8 to +37.3 cal K⁻¹ mol⁻¹, positive activation volumes from +18.2 to +30.0 cm³ mol⁻¹ and a linear free energy relationship of slope -1.09 ± 0.06 have been observed for a series of L including nitriles, ethers, amides and -PO donor ligands. It is concluded that a limiting dissociative, D, mechanism exists for the whole series of ligand exchange reactions studied.     

Interaction of vanadate with monosaccharides and nucleosides: a multinuclear NMR study

Proton, 13C and 51V nuclear magnetic resonance spectroscopy has been used to study the interaction of vanadate with several molecules containing more than one hydroxyl group, including various aldoses and nucleosides. The aldoses D-mannose and D-ribose mainly form tridentate complexes, of trigonal bipyramidal geometry, with vanadate at pH 7. These sugars use three consecutive hydroxyl groups, cis to each other, of their pyranose forms to bind vanadate in those cyclic triesters. Other aldoses, like D-glucose, which do not have this unique structural characteristic, do not form tridentate complexes, but can form weaker bidentate cyclic diesters using two consecutive pyranose cis hydroxyl groups. Of course, the pyranose forms of D-mannose and D-ribose, as well as the furanose form of D-ribose, also yield cyclic diesters of vanadate. All these aldoses form weak monodentate noncyclic monoesters of tetrahedral geometry using a single hydroxyl group. The nucleosides uridine, cytidine and adenosine form two complexes of trigonal bipyramidal geometry with vanadate. In these complexes, having 1:1 and 2:1 ligand-to-metal stoichiometries, the nucleosides form cyclic diesters with vanadate using their C2, and C3, hydroxyl groups.     

Amiodarone-liposome interaction: a multinuclear NMR and X-ray diffraction study

Amiodarone, a potent antiarrhythmic drug, is widely used in cardiology. Its electrophysiological effects, as well as many of its side effects, seem to involve lipids. We report here a multinuclear NMR and X-ray diffraction study of amiodarone in egg phosphatidylcholine liposomes and lipid multilayers. In proton NMR experiments, amiodarone alters the signal from the lipid trimethyl ammonium group for pH values ranging from 3.2 to 8.4; cholesterol does not cause this alteration. The addition of SCN- changes both the proton and phosphorus NMR spectra of liposomes containing amiodarone. For both proton and carbon NMR, amiodarone modifies the signal from the lipid methylene groups, but to a far lesser extent than does cholesterol. Incorporation of amiodarone in EPC bilayers also modifies the low-angle X-ray diffraction patterns, decreasing the lamellar repeat period at low water contents, but swelling the fluid spaces between bilayers at high water contents. Electron density profiles and modeling studies using the X-ray data indicate that amiodarone decreases the bilayer thickness and adds electron density at the interfacial region of the bilayer. Our analysis of the NMR and X-ray data indicates that the iodine atoms of amiodarone are located near the hydrocarbon/water interface and that the tertiary amine of amiodarone is in the headgroup region of the bilayer.     

Cytochrome c interactions with cardiolipin in bilayers: a multinuclear magic-angle spinning NMR study.

The influence of cytochrome c binding to cardiolipin bilayers on the motional characteristics of each component has been analyzed by magic-angle spinning (MAS) NMR. Observations were made by NMR of natural abundance 31P, 13C, and 1H nuclei in the lipid as well as sites enriched with 13C in the protein. Analysis of methyl carbons enriched in ([epsilon-13CH3]methionine)cytochrome c at residues 65 and 80 reveal quite different behavior for these sites when the protein was bound at a 1:15 molar ratio with hydrated cardiolipin. Cross-polarization (CP) shows a single broad resonance downfield in the methyl region which corresponds to the spectral characteristics of methionine 65 in the solution protein when subjected to moderate thermal perturbations. These observations suggest that although methionine 65 remains motionally restricted when the protein binds to the lipid bilayers, this residue becomes less shielded and exposed to more chemically distinct environments than in the native state of the protein. In contrast to its behavior in native oxidized protein, the methionine 80 methyl could be detected following direct pi/2 pulse excitation, and this residue is assumed to be released from the axial ligand site on the heme iron to become more exposed and highly mobile in the protein-lipid complex. An analysis of the CP response for natural abundance 13C nuclei in the lipid reveals a general increase in motions with slower rates (tens of kilohertz) on binding with cytochrome c, except for sites within the region of fatty acyl chain unsaturation which appear to be selectively mobilized in the complex with protein. It is concluded that, aside from effects on the unsaturated segments, the bound protein induces new modes of slow motions in the lipid assemblies rather than restricting the overall reorientation freedom of the lipid. The strong paramagnetic effects observed previously on the relaxation of phosphorus in protein-bound lipid [Spooner, P.J.R., & Watts, A. (1991) Biochemistry 30, 3880-3885] were not extended to any carbon and proton sites observable by MAS NMR in the lipid, and this infers a specific interaction of lipid phosphate groups with the heme. However, when protein was bound to cardiolipin mixed at a 1:4 mole ratio with dioleoylphosphatidylcholine in bilayers, no direct interaction with the heme was apparent from the phosphorus NMR relaxation behavior in this component, resolved by MAS. Instead, the spectral anisotropy of cardiolipin phosphorus was determined to be reduced, indicating that, on binding with cytochrome c, the headgroup organization was perturbed in this component.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR study of a Lewis(X) pentasaccharide derivative: solution structure and interaction with cations.

The structure and conformation of the synthetic pentasaccharide Gal(beta 1-4){Fuc(alpha 1-3)}GlcNAc(beta 1-3)Gal(beta 1-4)Glc-beta OMe of the Lewis(X) family has been determined by NMR spectroscopy in dimethyl sulfoxide and methanol. In these solvents, the binding constants with calcium have been evaluated as 9.5 and 29.6 M-1, respectively. Study of the interaction sites has been achieved through the use of paramagnetic divalent cations and distance triangulation methods. Two regions have been found, the first one in the vicinity of the fucose unit, the second one closer to the lactose part.     

A multinuclear NMR study of the interactions of cations with proteoglycans, heparin, and Ficoll

Measurements of NMR relaxation rates of 23Na, 39K, 25Mg and 43Ca were used to evaluate the extent of cation binding in solution to bovine nasal cartilage proteoglycans, hog mucosal heparin, and Ficoll (Pharmacia). The two most important factors determining relaxation rates in the presence of the polymers examined were polymer concentration and charge density. We found that proteoglycans did not bind monovalent cations but did bind divalent cations to a relatively small extent. Heparin bound monovalent and divalent cations to a much greater extent. Assembly of glycosaminoglycan chains into proteoglycan aggregates had no effect on the extent of cation binding.     

Beta-adrenergic stimulation of C6 glioma cells: effects of cAMP overproduction on cellular metabolites. A multinuclear NMR study.

We used 31P-NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a beta-adrenergic agonist, isoproterenol. In the presence of 3-isobutyl-1-methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20-25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P-NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase). 13C-NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1-13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C-NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H-NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentose-phosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to beta-adrenergic stimulation and cAMP overproduction.     

Competition between Li+ and Mg2+ for ATP and ADP in aqueous solution: a multinuclear NMR study

We used 7Li NMR spin-lattice relaxation times and 31P NMR chemical shifts to study the binding of Li+ and Mg2+ to the phosphate moieties of ATP and ADP. To examine the binding of Li+ and Mg2+ to the base and ribose moieties, we used 1H and 13C NMR chemical shifts. The 7Li NMR relaxation times of Li+/Mg2+ mixtures of ATP or ADP increased with increasing concentrations of Mg2+, suggesting competition between the two ions for adenine nucleotides. No significant binding of Li+ and Mg2+ to the base and ribose moieties occurred. At the pH and ionic strength used, 2:1 and 1:1 species of the Li(+)-ATP and Li+-ADP complexes were present, with the 2:1 species predominating. In contrast, 1:1 species predominated for the Mg(2+)-ADP and Mg(2+)-ATP complexes. We calculated the Li(+)-nucleotide binding constants in the presence and absence of Mg2+ and found them to be somewhat greater in the presence of Mg2+. Although competition between Li+ and Mg2+ for ATP and ADP phosphate binding sites in solution is consistent with the 31P chemical shift data, the possibility that the Li+ and Mg2+ form mixed complexes with the phosphate groups of ATP or ADP cannot be ruled out.     

Structural differences in the two calcium binding sites of the porcine intestinal calcium binding protein: a multinuclear NMR study

Cadmium-113 and calcium-43 NMR spectra of Cd2+ and Ca2+ bound to the porcine intestinal calcium binding protein (ICaBP; Mr 9000) contain two resonances. The first resonance is characterized by NMR parameters resembling those found for these cations bound to proteins containing the typical helix-loop-helix calcium binding domains of parvalbumin, calmodulin, and troponin C, which are defined as EF-hands by Kretsinger [Kretsinger, R. H. (1976) Annu. Rev. Biochem. 45, 239]. The second resonance in both spectra has a unique chemical shift and is consequently assigned to the metal ion bound in the N-terminal site of ICaBP. This site is characterized by an insertion of a proline in the loop of the helix-loop-helix domain and will be called the pseudo-EF-hand site. The binding of Cd2+ to the apo form of ICaBP is sequential. The EF-hand site is filled first. Both binding sites have similar, but not identical, affinities for Ca2+: at a Ca2+ to protein ratio of 1:1, 65% of the ion is bound in the EF-hand site and 35% in the pseudo-EF-hand site. The two sites do not appear to act independently; thus, replacement of Ca2+ or Cd2+ by La3+ in the EF-hand site causes changes in the environment of the ions in the pseudo-EF-hand site. In addition, the chemical shift of Cd2+ bound to the EF-hand site is dependent on the presence or absence of Ca2+ or Cd2+ in the pseudo-EF-hand site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific interactions of sticholysin I with model membranes: An NMR study

Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N‐terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by ¹H‐NMR the environment of these regions in water and in the presence of membrane‐mimicking micelles. Unlike other peptides from homologous actinoporins, the synthetic peptide corresponding to residues 1–30 tends to form helix in water and is more helical in either trifluoroethanol or dodecylphosphocholine (DPC) micelles. In these environments, it forms a helix‐turn‐helix motif with the last α‐helical segment matching the native helix‐α₁ (residues 14–24) present in the complete protein. The first helix (residues 4–9) is less populated and is not present in the water‐soluble protein structure. The characterization of wild‐type StnI structure in micelles shows that the helix‐α₁ is maintained in its native structure and that this micellar environment does not provoke its detachment from the protein core. Finally, the study of the aromatic resonances has shown that the motional flexibility of specific rings is perturbed in the presence of micelles. On these bases, the implication of the aromatic rings of Trp‐111, Tyr‐112, Trp‐115, Tyr‐132, Tyr‐136, and Tyr‐137, in the interaction between StnI and the micelle is discussed. Based on all the findings, a revised model for StnI interaction with membranes is proposed, which accounts for differences in its behavior as compared with other highly homologous sticholysins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

A multinuclear NMR study of the active site of an endoglucanase from a strain of Bacillus. Use of Trp residues as structural probes.

In the hydrolytic reaction catalyzed by an endoglucanase from a Bacillus strain (endoglucanase K), 2 of 12 Trp residues, Trp174 and Trp243, are responsible for binding of the substrate and/or for the catalysis (Kawaminami, S., Ozaki, K., Sumitomo, N., Hayashi, Y., Ito, S., Shimada, I., and Arata, Y. (1994) J. Biol. Chem. 269, 28752-28756). Here we report results of a stable isotope-aided NMR analysis of the active site of endoglucanase K, using Trp174 and Trp243 as structural probes. Hydrogen-deuterium exchange experiments performed for the NH protons of main and side chains of Trp residues revealed that Trp174 and Trp243 are located in the hydrophilic and hydrophobic microenvironments in the active site, respectively. We also carried out pH titration experiments for indole C2 proton resonances of Trp residues and measured the pH dependence of specific activities for wild-type endoglucanase K and its mutants in which Glu or Asp residues are replaced with their respective amide forms. On the basis of the results obtained from the present study, we conclude that (a) Glu130 and Asp191, which are in spatial proximity to Trp174 and Trp243 in the active site, play a crucial role in the enzymatic activity; (b) Glu130 and Asp191 interact with each other in the active site, leading to an increase in the pKa values to 5.5 for both amino acid residues; and (c) the pKa values of Glu130 and Asp191 would lead to an unusually narrow pH-activity profile of the endoglucanase K.     

Changes in organic solutes,volume, energy state,and metabolism associated with osmotic stress in a glial cell line: A multinuclear NMR study

Diffusion-weighted in vivo¹H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM.¹H-,¹³C-and³¹P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo³¹P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.To whom to address reprint requests.     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (²H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogens in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T₁ relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

Structure of the K-turn U4 RNA: a combined NMR and SANS study

K-turn motifs are universal RNA structural elements providing a binding platform for proteins in several cellular contexts. Their characteristic is a sharp kink in the phosphate backbone that puts the two helical stems of the protein-bound RNA at an angle of 60°. However, to date no high-resolution structure of a naked K-turn motif is available. Here, we present the first structural investigation at atomic resolution of an unbound K-turn RNA (the spliceosomal U4-Kt RNA) by a combination of NMR and small-angle neutron scattering data. With this study, we wish to address the question whether the K-turn structural motif assumes the sharply kinked conformation in the absence of protein binders and divalent cations. Previous studies have addressed this question by fluorescence resonance energy transfer, biochemical assays and molecular dynamics simulations, suggesting that the K-turn RNAs exist in equilibrium between a kinked conformation, which is competent for protein binding, and a more extended conformation, with the population distribution depending on the concentration of divalent cations. Our data shows that the U4-Kt RNA predominantly assumes the more extended conformation in the absence of proteins and divalent cations. The internal loop region is well structured but adopts a different conformation from the one observed in complex with proteins. Our data suggests that the K-turn consensus sequence does not per se code for the kinked conformation; instead the sharp backbone kink requires to be stabilized by protein binders.     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (2H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogen in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T1 relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modelling methods. Part I: Studies in water.

Arginine vasopressin (AVP) and mesotocin (MT) belong to the neurohypophyseal hormone family. The former plays a very important role in the control of urine concentration and the blood pressure in mammals, whereas the latter stimulates uterine concentration and initiates birth in amphibians, marsupials, wallabies, birds, and fishes. Analysis of their 3D structure could be helpful for understanding the evolutionary relationship between all vasopressin- and oxytocin-like hormones. In addition, it allows design of new analogs with appropriate biological activity for humans and animals. In this paper, we present the conformational studies of AVP and MT, under the aqueous conditions. In our investigations, we used 2D NMR spectroscopy and time-averaged molecular dynamics calculations in explicit water. Our studies have shown that both peptides, despite displaying a high sequence homology, differ from each other with regard to the three-dimensional structure. They are in conformational equilibrium as a result of the cis/trans isomerization across the Cys(6)-Pro(7) peptide bond. Both peptides form beta-turns in their cyclic part, wherein the C-terminal fragment of MT is bent, whereas that of AVP is extended.