Two-dimensional 13C-1H heteronuclear correlation NMR spectroscopic studies for the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with a new Helicobacter pylori eradicating agent (TG44) in the amorphous state

The interaction of a newly developed Helicobacter pylori eradicating agent (TG44, 4-methylbenzyl-4'-[trans-4-(guanidinomethyl)cyclohexylcarbonyloxy]-biphenyl-4-carboxlylate monohydrochloride) with cyclomaltoheptaose (beta-cyclodextrin, beta-CyD) in the solid state was studied by high-speed frequency-switched Lee-Goldburg (FSLG) (13)C-(1)H heteronuclear correlation (HETCOR) NMR experiments. The TG44/beta-CyD solid complex in a 1:1 stoichiometry was prepared by the grinding method. Powder X-ray diffractometry confirmed that the complex is in an amorphous state. The solid-state (13)C signals of TG44 and beta-CyD were significantly broadened by the complexation. As the temperature increased, the (13)C signals of the aromatic moieties of TG44 were insignificantly influenced, whereas those of the cyclohexyl moiety became sharper. The T1(rho) H values of the aromatic moieties of TG44 were almost the same as those of the beta-CyD carbons, whereas those of other TG44 carbons gave much smaller values. The (13)C-(1)H HETCOR spectra gave the intermolecular correlation peaks between the aromatic carbons of TG44 and the beta-CyD protons or between the biphenyl protons of TG44 and the beta-CyD carbons, when measured using longer contact times (500 and 1500mus). On the basis of these solid NMR spectroscopic data together with aqueous NMR data, we assume that beta-CyD includes predominantly the biphenyl moiety of TG44 in the solid state. (13)C-(1)H HETCOR spectroscopy is particularly useful for the determination of inclusion modes of the complexes that occurring in an amorphous form.     

New cyclomaltoheptaose (beta-cyclodextrin) derivative 2-O-(2-hydroxybutyl)cyclomaltoheptaose: preparation and its application for the separation of enantiomers of drugs by capillary electrophoresis

A new soluble cyclomaltoheptaose (cyclodextrin) derivative, 2-O-(2-hydroxybutyl)cyclomaltoheptaose [2-O-(2-hydroxybutyl)-beta-cyclodextrin, 2-HB-beta-CD], was prepared and studied as an efficient chiral selector in the separation of racemic mixtures of drugs by capillary electrophoresis (CE). Results showed that 2-HB-beta-CD could provide higher separating capability than that of beta-CD and the similarly substituted 2-HP-beta-CD.     

The use of spin desalting columns in DMSO‐quenched H/D‐exchange NMR experiments

Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D₂O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D₂O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.     

Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains V(H) and V(L). Both V(H) and V(L) adopt essentially native-like exchange protection within the dead time of the manual-mixing H/D exchange experiment (10 s) and in the case of V(L), which contains two cis-prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than V(L) and V(H). Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in V(L), this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association.     

Imazalil-cyclomaltoheptaose (beta-cyclodextrin) inclusion complex: preparation by supercritical carbon dioxide and 13C CPMAS and 1H NMR characterization

An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H).     

Thermodynamic stability measurements on multimeric proteins using a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based method

We recently reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins. In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material. Here we report on the application of SUPREX to the analysis of multimeric protein systems. Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V(a)L(d) trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20. As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems. Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopy-based methods.     

Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase A

The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (R(I)alpha(94-244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free R(I)alpha(94-244), which likely represents newly synthesized protein, (2) R(I)alpha(94-244) bound to the catalytic (C) subunit, or holoenzyme, and (3) R(I)alpha(94-244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site.     

Kinetic studies on the first dihydrogen aquacomplex, [Ru(H2)(H2O)5]: Formation under H2 pressure and catalytic H/D isotope exchange in water

The ruthenium(II) hexaaqua complex [Ru(H₂O)₆]²⁺ reacts with dihydrogen under pressure to give the η²-dihydrogen ruthenium(II) pentaaqua complex [Ru(H₂)(H₂O)₅]²⁺.The complex was characterized by ¹H, ²H and ¹⁷O NMR: δ_H = −7.65 ppm, J_HD = 31.2 Hz, δ_O = −80.4 ppm (trans to H₂) and δ_O = −177.4 ppm (cis to H₂).The H-H distance in coordinated dihydrogen was estimated to 0.889 Å from J_HD, which is close to the value obtained from DFT calculations (0.940 Å).Kinetic studies were performed by ¹H and ²H NMR as well as by UV-Vis spectroscopy, yielding the complex formation rate and equilibrium constants: k_f = (1.7 ± 0.2) × 10⁻³ kg mol⁻¹ s⁻¹ and K_eq = 4.0 ± 0.5 mol kg⁻¹.The complex formation rate with dihydrogen is close to values reported for other ligands and thus it is assumed that the reaction with dihydrogen follows the same mechanisn (I_d).In deuterated water, one can observe that [Ru(H₂)(H₂O)₅]²⁺ catalyses the hydrogen exchange between the solvent and the dissolved dihydrogen.A hydride is proposed as the intermediate for this exchange.Using isotope labeling, the rate constant for the hydrogen exchange on the η²-dihydrogen ligand was determined as k₁ = (0.24 ± 0.04) × 10⁻³ s⁻¹.The upper and lower limits of the pK_a of the coordinated dihydrogen ligand have been estimated:3 < pK_a < 14.     

Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): Effect of parathyroid hormone (PTH)

Summary Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10^–8 m; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10^–8 m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10^–8 m) resulted in a rapid and transient increase in [Ca²⁺] _i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca²⁺ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/P _i cotransport.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation and the Geigy-Jubiläumsstiftung. We are grateful to Denise Rossi and Christa Knellwolf for their excellent secretarial assistance.     

Polymorphic triple beta-sheet structures contribute to amide hydrogen/deuterium (H/D) exchange protection in the Alzheimer amyloid beta42 peptide

Characterization of the polymorphic structural range of Aβ oligomers is important to the understanding of the mechanisms of toxicity. Yet for highly polymorphic ensembles, experimental structural elucidation is difficult. Here, we use a combination of NMR solvent protection experiments and computational structural screening to identify major species in the amyloid conformational ensemble. We examined the polymorphic pentamer and fibril seeds of Aβ42 and its mutants and compared the theoretical backbone amide protection obtained from simulations with experimental hydrogen/deuterium (H/D) exchange protection ratio. We observed that highly flexible pentamers do not share structural similarities with fibril seed oligomers, except the turn regions. We found that a novel amyloid structural motif of a triple β-sheet, with the N-terminal residues interacting with the core (Lys(17)-Glu(22)) β-sheet region, correlates with H/D exchange protection. The triple β-sheet Aβ42 oligomer has a minimal exposure of hydrophobic residues and is further stabilized by the E22Q (Dutch) mutation in Alzheimer disease. The experimental H/D exchange solvent protection ratio implies that triple β-sheet fibrils and globulomers could coexist in the Aβ42 ensemble, pointing to a broad heterogeneous aggregate population. Our results suggest that an approach that combines computational modeling with NMR protection data can be a useful strategy for obtaining clues to the preferred conformational species of the assemblies in solution and help in alleviating experimental difficulties and consequently possible errors in the exchange data for Aβ42 fibrils.     

Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFbeta)

Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSFbeta), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange-in shows that in solution the conserved four-helix bundle of (rhM-CSFbeta) has fast and moderately fast exchangeable sections of amide hydrogens in the alphaA helix, and mostly slow exchanging sections of amide hydrogens in the alphaB, alphaC, and alphaD helices. Most of the amide hydrogens in the loop between the beta1 and beta4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X-ray crystallographic data for rhM-CSFalpha than with the average B-factor. The rates of H/D exchange in rhM-CSFbeta appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the alphaD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150-221 present in rhM-CSFbeta, but not rhM-CSFalpha, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the alphaD helix is due to interaction of the carboxy-terminal tail with this helical segment.     

Prevalence of C282Y and H63D mutations in the haemochromatosis (HFE) gene in Tunisian population

The studies of the HFE mutations: H63D and C282Y in North African populations have revealed the extreme rarity or even the absence of the C282Y mutation. We have examined 1140 chromosomes (570 Tunisian people) for the presence of the two HFE mutations by PCR-RFLP analysis. We have found that the allele frequencies are, respectively, 15.17% (+/-2.1%) for the H63D and 0.09% (+/-0.17%) for the C282Y. These results are consistent with the worldwide spread of the H63D mutation and the north European restriction of the C282Y. This study will be completed by determining whether homozygote trait for H63D and associated risk factors (beta thalassémia) can lead to iron overload in Tunisia.     

Synthesis of some substituted pyrazinopyridoindoles and 3D QSAR studies along with related compounds: piperazines, piperidines, pyrazinoisoquinolines, and diphenhydramine, and its semi-rigid analogs as antihistamines (H1)

3D QSAR studies on the title compounds led to the development of a model with three biophoric sites and six secondary sites viz. H-acceptor (ACC), H-donor (DON), heteroatom (presence), hydrophobic (hydrophobicity), steric (refractivity), and a ring (presence) along with total hydrophobicity and total refractivity as global properties. The model predicted the test set of compounds reasonably well. Three of the five newly synthesized 2-substituted octahydropyrazinopyridoindoles have shown potent antihistaminic H₁ activity with less toxicity and sedation potential.     

R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry

The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/²H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.     

Biosynthesis of the sesquiterpene germacrene D in Solidago canadensis: 13C and (2)H labeling studies

The biogenetic origin of the isoprenoid building blocks of the sesquiterpene germacrene D was studied in Solidago canadensis. Feeding experiments were carried out with 1-[5,5-D(2)]deoxy-D-xylulose-5-phosphate (D(2)-DOXP), [5-13C]mevalonolactone (13C-MVL) and [1-13C]-D-glucose. The hydrodistillate of a cut shoot fed with D(2)-DOXP was investigated by enantio-MDGC-MS and the volatile fraction of a shoot supplied with 13C-MVL was examined by GC-C-IRMS. The incorporation of [1-13C]-D-glucose was analyzed by quantitative 13C NMR spectroscopy after isolation of germacrene D from the essential oil. Our labeling studies revealed that the biosynthesis of the C-15 skeleton of sesquiterpene germacrene D in Solidago canadensis proceeds predominantly via the methylerythritol phosphate pathway.     

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP⁺ oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron?sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.     

A pyridine adduct of bis(di-iso-butyldithiocarbamato-S,S′)cadmium(II): Multinuclear (C, N, Cd) CP/MAS NMR spectroscopy, crystal and molecular structure, and thermal behaviour

Crystalline bis(N,N-di-iso-butyldithiocarbamato-S,S′)(pyridine)cadmium(II) - adduct 1 was prepared and studied by means of multinuclear ¹³C, ¹⁵N, ¹¹³Cd CP/MAS NMR spectroscopy, single-crystal X-ray diffraction and simultaneous thermal analysis (STA). In molecular structure 1, the cadmium atom coordinates with four sulphur atoms and one nitrogen atom of pyridine, forming a coordination polyhedron [CdS₄N], whose geometry is an almost ideal tetragonal pyramidal (C_4v). The coordinated py molecule is in the apical position, while two structurally non-equivalent di-iso-butyldithiocarbamate ligands, playing the same terminal S,S′-chelating function, define the basal plane. To characterise additionally the structural state of the cadmium atom in this fivefold coordination, ¹¹³Cd chemical shift anisotropy (CSA) parameters, δ_aniso and η, were calculated from experimental MAS NMR spectra that revealed an almost axially symmetric ¹¹³Cd chemical shift tensor. From a combination of TG and DSC measurements taken under an argon atmosphere, we found that the mass of adduct 1 is lost in two steps involving initial desorption of coordinated py molecules with subsequent thermal destruction of liberated cadmium(II) di-iso-butyldithiocarbamate, with yellow-orange, fine-powdered solid CdS as the final product.