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Sample records for amide hydrogen exchange

  1. Hydrogen bond stabilities in membrane-reconstituted alamethicin from amide-resolved hydrogen-exchange measurements.

    Science.gov (United States)

    Dempsey, C E; Handcock, L J

    1996-01-01

    Amide-resolved hydrogen-deuterium exchange-rate constants were measured for backbone amides of alamethicin reconstituted in dioleoylphosphatidylcholine vesicles by an exchange-trapping method combined with high-resolution nuclear magnetic resonance spectroscopy. In vesicles containing alamethicin at molar ratios between 1:20 and 1:100 relative to lipid, the exchange-rate constants increased with increasing volume of the D20 buffer in which the vesicles were suspended, indicating that exchange under these conditions is dominated by partitioning of the peptide into the aqueous phase. This was supported by observation of a linear relationship between the exchange-rate constants for amides in membrane-reconstituted alamethicin and those for amides in alamethicin dissolved directly into D2O buffer. Significant protection of amides from exchange with D2O buffer in membrane-reconstituted alamethicin is interpreted in terms of stabilization by helical hydrogen bonding. Under conditions in which amide exchange occurred by partitioning of the peptide into solution, only lower limits for hydrogen-bond stabilities in the membrane were determined; all the potentially hydrogen-bonded amides of alamethicin are at least 1000-fold exchange protected in the membrane-bound state. When partitioning of alamethicin into the aqueous phase was suppressed by hydration of reconstituted vesicles in a limiting volume of water [D2O:dioleoylphosphatidylcholine:alamethicin; 220:1:0.05; (M:M:M)], the exchange-protection factors exhibited helical periodicity with highly exchange-protected, and less well-protected, amides on the nonpolar and polar helix faces, respectively. The exchange data indicate that, under the conditions studied, alamethicin adopts a stable helical structure in DOPC bilayers in which all the potentially hydrogen-bonded amides are stabilized by helical hydrogen bonds. The protection factors define the orientation of the peptide helix with respect to an aqueous phase, which is

  2. Hydrogen bond stabilities in membrane-reconstituted alamethicin from amide-resolved hydrogen-exchange measurements.

    Science.gov (United States)

    Dempsey, C E; Handcock, L J

    1996-04-01

    Amide-resolved hydrogen-deuterium exchange-rate constants were measured for backbone amides of alamethicin reconstituted in dioleoylphosphatidylcholine vesicles by an exchange-trapping method combined with high-resolution nuclear magnetic resonance spectroscopy. In vesicles containing alamethicin at molar ratios between 1:20 and 1:100 relative to lipid, the exchange-rate constants increased with increasing volume of the D20 buffer in which the vesicles were suspended, indicating that exchange under these conditions is dominated by partitioning of the peptide into the aqueous phase. This was supported by observation of a linear relationship between the exchange-rate constants for amides in membrane-reconstituted alamethicin and those for amides in alamethicin dissolved directly into D2O buffer. Significant protection of amides from exchange with D2O buffer in membrane-reconstituted alamethicin is interpreted in terms of stabilization by helical hydrogen bonding. Under conditions in which amide exchange occurred by partitioning of the peptide into solution, only lower limits for hydrogen-bond stabilities in the membrane were determined; all the potentially hydrogen-bonded amides of alamethicin are at least 1000-fold exchange protected in the membrane-bound state. When partitioning of alamethicin into the aqueous phase was suppressed by hydration of reconstituted vesicles in a limiting volume of water [D2O:dioleoylphosphatidylcholine:alamethicin; 220:1:0.05; (M:M:M)], the exchange-protection factors exhibited helical periodicity with highly exchange-protected, and less well-protected, amides on the nonpolar and polar helix faces, respectively. The exchange data indicate that, under the conditions studied, alamethicin adopts a stable helical structure in DOPC bilayers in which all the potentially hydrogen-bonded amides are stabilized by helical hydrogen bonds. The protection factors define the orientation of the peptide helix with respect to an aqueous phase, which is

  3. Dynamics of urokinase receptor interaction with Peptide antagonists studied by amide hydrogen exchange and mass spectrometry

    DEFF Research Database (Denmark)

    Jørgensen, Thomas J D; Gårdsvoll, Henrik; Danø, Keld

    2004-01-01

    Using amide hydrogen exchange combined with electrospray ionization mass spectrometry, we have in this study determined the number of amide hydrogens on several peptides that become solvent-inaccessible as a result of their high-affinity interaction with the urokinase-type plasminogen activator...... receptor (uPAR). These experiments reveal that at least six out of eight amide hydrogens in a synthetic nine-mer peptide antagonist (AE105) become sequestered upon engagement in uPAR binding. Various uPAR mutants with decreased affinity for this peptide antagonist gave similar results, thereby indicating...... that deletion of the favorable interactions involving the side chains of these residues in uPAR does not affect the number of hydrogen bonds established by the main chain of the peptide ligand. The isolated growth factor-like domain (GFD) of the cognate serine protease ligand for uPAR showed 11 protected amide...

  4. Amide hydrogen-deuterium exchange kinetics as a test of structural states of proteins

    International Nuclear Information System (INIS)

    Popescu, Aurel I.; Craescu, Constantin

    2000-01-01

    The amide hydrogen-deuterium exchange (AHD EX ) method gives information about the accessibility degree of amide protons, on their interaction with the physico-chemical microenvironment, and implicitly, on the 3D structure of proteins. This method as useful both for the characterisation of the native state and of conformational modifications induced by protein interaction with different physico-chemical agents. The rate of the amide proton exchange for deuterium can be measured by various physical methods (NMR, MS, pulsed H/D exchange, etc.) evaluating the so-called protection factor, P, defined as the ratio between the exchange constant, k RC , experimentally determined on model peptides in a random coil conformation and the actual exchange constant, k EX , derived from the NMR spectra (e.g. the heteronuclear multiple-quantum correlation spectroscopy, HMQC). In this work, the NMR spectra of the immunophilin domain of FKBP59-I protein (both in unligated state and when it is ligated with FK506 immunosuppressor) recorded at corrected pH = 7.2, T= 308 K, are interpreted. The results show a large variety of kinetic constant values, spanning from those of very rapidly exchanging protons (non-protected) to those of very slow exchanging once (highly protected). After binding FK506, the protection factor of FKBP59-I amide protons, are significantly increased. (authors)

  5. Amide proton solvent protection in amylin fibrils probed by quenched hydrogen exchange NMR.

    Directory of Open Access Journals (Sweden)

    Andrei T Alexandrescu

    Full Text Available Amylin is an endocrine hormone that accumulates in amyloid plaques in patients with advanced type 2 diabetes. The amyloid plaques have been implicated in the destruction of pancreatic β-cells, which synthesize amylin and insulin. To better characterize the secondary structure of amylin in amyloid fibrils we assigned the NMR spectrum of the unfolded state in 95% DMSO and used a quenched hydrogen-deuterium exchange technique to look at amide proton solvent protection in the fibrils. In this technique, partially exchanged fibrils are dissolved in 95% DMSO and information about amide proton occupancy in the fibrils is determined from DMSO-denatured monomers. Hydrogen exchange lifetimes at pH 7.6 and 37°C vary between ∼5 h for the unstructured N-terminus to 600 h for amide protons in the two β-strands that form inter-molecular hydrogen bonds between amylin monomers along the length of the fibril. Based on the protection data we conclude that residues A8-H18 and I26-Y37 comprise the two β-strands in amylin fibrils. There is variation in protection within the β-strands, particularly for strand β1 where only residues F15-H18 are strongly protected. Differences in protection appear to be due to restrictions on backbone dynamics imposed by the packing of two-layers of C2-symmetry-related β-hairpins in the protofilament structure, with strand β1 positioned on the surface and β2 in the interior.

  6. NMR Analysis of Amide Hydrogen Exchange Rates in a Pentapeptide-Repeat Protein from A. thaliana.

    Science.gov (United States)

    Xu, Shenyuan; Ni, Shuisong; Kennedy, Michael A

    2017-05-23

    At2g44920 from Arabidopsis thaliana is a pentapeptide-repeat protein (PRP) composed of 25 repeats capped by N- and C-terminal α-helices. PRP structures are dominated by four-sided right-handed β-helices typically consisting of mixtures of type II and type IV β-turns. PRPs adopt repeated five-residue (Rfr) folds with an Rfr consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Unlike other PRPs, At2g44920 consists exclusively of type II β-turns. At2g44920 is predicted to be located in the thylakoid lumen although its biochemical function remains unknown. Given its unusual structure, we investigated the biophysical properties of At2g44920 as a representative of the β-helix family to determine if it had exceptional global stability, backbone dynamics, or amide hydrogen exchange rates. Circular dichroism measurements yielded a melting point of 62.8°C, indicating unexceptional global thermal stability. Nuclear spin relaxation measurements indicated that the Rfr-fold core was rigid with order parameters ranging from 0.7 to 0.9. At2g44920 exhibited a striking range of amide hydrogen exchange rates spanning 10 orders of magnitude, with lifetimes ranging from minutes to several months. A weak correlation was found among hydrogen exchange rates, hydrogen bonding energies, and amino acid solvent-accessible areas. Analysis of contributions from fast (approximately picosecond to nanosecond) backbone dynamics to amide hydrogen exchange rates revealed that the average order parameter of amides undergoing fast exchange was significantly smaller compared to those undergoing slow exchange. Importantly, the activation energies for amide hydrogen exchange were found to be generally higher for the slowest exchanging amides in the central Rfr coil and decreased toward the terminal coils. This could be explained by assuming that the concerted motions of two preceding or following coils required for hydrogen bond disruption and amide hydrogen exchange have a higher activation energy

  7. Hydrogen exchange

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Rand, Kasper Dyrberg

    2016-01-01

    Hydrogen exchange (HX) monitored by mass spectrometry (MS) is a powerful analytical method for investigation of protein conformation and dynamics. HX-MS monitors isotopic exchange of hydrogen in protein backbone amides and thus serves as a sensitive method for probing protein conformation...... and dynamics along the entire protein backbone. This chapter describes the exchange of backbone amide hydrogen which is highly quenchable as it is strongly dependent on the pH and temperature. The HX rates of backbone amide hydrogen are sensitive and very useful probes of protein conformation......, as they are distributed along the polypeptide backbone and form the fundamental hydrogen-bonding networks of basic secondary structure. The effect of pressure on HX in unstructured polypeptides (poly-dl-lysine and oxidatively unfolded ribonuclease A) and native folded proteins (lysozyme and ribonuclease A) was evaluated...

  8. Conformational analysis of Epac activation using amide hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Brock, Melissa; Fan, Fenghui; Mei, Fang C; Li, Sheng; Gessner, Christopher; Woods, Virgil L; Cheng, Xiaodong

    2007-11-02

    Exchange proteins directly activated by cAMP (Epac) play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Rap. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen/deuterium exchange and structural modeling. Our studies show that cAMP induces significant conformational changes that lead to a spatial rearrangement of the regulatory components of Epac and allows the exposure of the catalytic core for effector binding without imposing significant conformational change on the catalytic core. Homology modeling and comparative structural analyses of the cAMP binding domains of Epac and cAMP-dependent protein kinase (PKA) lead to a model of Epac activation, in which Epac and PKA activation by cAMP employs the same underlying principle, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different.

  9. Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Hofmann, Tommy; Samsonov, Sergey A; Pichert, Annelie; Lemmnitzer, Katharina; Schiller, Jürgen; Huster, Daniel; Pisabarro, M Teresa; von Bergen, Martin; Kalkhof, Stefan

    2015-11-01

    The recruitment of different chemokines and growth factors by glycosaminoglycans (GAGs) such as chondroitin sulfate or hyaluronan plays a critical role in wound healing processes. Thus, there is a special interest in the design of artificial extracellular matrices with improved properties concerning GAG interaction with common regulating proteins. In this study, amide hydrogen/deuterium (H/D) exchange mass spectrometry (HDX MS) combined with molecular modeling and docking experiments was used to obtain structural models of proinflammatory chemokine interleukin-8 (IL-8) in complex with hexameric chondroitin sulfate. Experiments on the intact protein showed a difference in deuterium labeling of IL-8 due to chondroitin sulfate binding. The extent of deuteration was reduced from 24% to 13% after 2 min exchange time, which corresponds to a reduced exchange of approximately 10 backbone amides. By local HDX MS experiments, H/D exchange information on the complete sequence of IL-8 could be obtained. A significantly reduced H/D exchange, especially of the C-terminal α-helical region comprising amino acids 70-77 and to the loop comprising amino acids 27-29 was observed in the presence of chondroitin sulfate. HDX MS data were used to model the IL-8/chondroitin sulfate complex. The binding interface of IL-8 and chondroitin sulfate determined this way correlated excellently with the corresponding NMR based atomistic model previously published. Our results demonstrate that HDX-MS in combination with molecular modeling is a valuable approach for the analysis of protein/GAG complexes at physiological pH, temperature, and salt concentration. The fact that HDX-MS requires only micrograms of protein and GAGs makes it a very promising technique to address protein-GAG interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data.

    Science.gov (United States)

    Hamuro, Yoshitomo; E, Sook Yen

    2018-03-02

    The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. Graphical Abstract ᅟ.

  11. Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data

    Science.gov (United States)

    Hamuro, Yoshitomo; E, Sook Yen

    2018-03-01

    The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. [Figure not available: see fulltext.

  12. Subzero temperature chromatography for reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Venable, John D; Okach, Linda; Agarwalla, Sanjay; Brock, Ansgar

    2012-11-06

    Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.

  13. pH and urea dependence of amide hydrogen-deuterium exchange rates in the beta-trefoil protein hisactophilin.

    Science.gov (United States)

    Houliston, R Scott; Liu, Chengsong; Singh, Laila M R; Meiering, Elizabeth M

    2002-01-29

    Amide hydrogen/deuterium exchange rates were measured as a function of pH and urea for 37 slowly exchanging amides in the beta-trefoil protein hisactophilin. The rank order of exchange rates is generally maintained under different solution conditions, and trends in the pH and urea dependence of exchange rates are correlated with the rank order of exchange rates. The observed trends are consistent with the expected behavior for exchange of different amides via global and/or local unfolding. Analysis of the pH dependence of exchange in terms of rate constants for structural opening and closing reveals a wide range of rates in different parts of the hisactophilin structure. The slowest exchanging amides have the slowest opening and closing rates. Many of the slowest exchanging amides are located in trefoil 2, but there are also some slow exchanging amides in trefoils 1 and 3. Slow exchangers tend to be near the interface between the beta-barrel and the beta-hairpin triplet portions of this single-domain structure. The pattern of exchange behaviour in hisactophilin is similar to that observed previously in interleukin-1 beta, indicating that exchange properties may be conserved among beta-trefoil proteins. Comparisons of opening and closing rates in hisactophilin with rates obtained for other proteins reveal clear trends for opening rates; however, trends in closing rates are less apparent, perhaps due to inaccuracies in the values used for intrinsic exchange rates in the data fitting. On the basis of the pH and urea dependence of exchange rates and optical measurements of stability and folding, EX2 is the main exchange mechanism in hisactophilin, but there is also evidence for varying levels of EX1 exchange at low and high pH and high urea concentrations. Equilibrium intermediates in which subglobal portions of structure are cooperatively disrupted are not apparent from analysis of the urea dependence of exchange rates. There is, however, a strong correlation between

  14. Thermal stability of human α-crystallins sensed by amide hydrogen exchange

    Science.gov (United States)

    Hasan, Azeem; Yu, Jiong; Smith, David L.; Smith, Jean B.

    2004-01-01

    The α-crystallins, αA and αB, are major lens structural proteins with chaperone-like activity and sequence homology to small heat-shock proteins. As yet, their crystal structures have not been determined because of the large size and heterogeneity of the assemblies they form in solution. Because α-crystallin chaperone activity increases with temperature, understanding structural changes of α-crystallin as it is heated may help elucidate the mechanism of chaperone activity. Although a variety of techniques have been used to probe changes in heat-stressed α-crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens α-crystallin using hydrogen/deuterium exchange in conjunction with enzymatic digestion and analysis by mass spectrometry. This technique has the advantage of sensing structural changes along much of the protein backbone and being able to detect changes specific to αA and αB in the native assembly. The reactivity of the amide linkages to hydrogen/deuterium exchange was determined for 92% of the sequence of αA and 99% of αB. The behavior of αA and αB is remarkably similar. At low temperatures, there are regions at the beginning of the α-crystallin domains in both αA and αB that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of αA also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in αA 72–75 and αB 76–79, two regions considered critical for chaperone activity. PMID:14739319

  15. Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange

    DEFF Research Database (Denmark)

    Rist, Wolfgang; Jørgensen, Thomas J D; Roepstorff, Peter

    2003-01-01

    Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock...... gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37...... promoters. The correlated exchange is shown to constitute a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy. We propose that this gradual decrease in stabilization energy of domain sigma 2 with increasing temperatures facilitates...

  16. Sub-Zero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen Deuterium Exchange Mass Spectrometry

    OpenAIRE

    Venable, John D.; Okach, Linda; Agarwalla, Sanjay; Brock, Ansgar

    2012-01-01

    Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises fr...

  17. Automated Hydrogen/Deuterium Exchange Electron Transfer Dissociation High Resolution Mass Spectrometry Measured at Single-Amide Resolution

    OpenAIRE

    Landgraf, Rachelle R.; Chalmers, Michael J.; Griffin, Patrick R.

    2011-01-01

    Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer....

  18. NMR-Based Amide Hydrogen-Deuterium Exchange Measurements for Complex Membrane Proteins: Development and Critical Evaluation

    Science.gov (United States)

    Czerski, Lech; Vinogradova, Olga; Sanders, Charles R.

    2000-01-01

    A method for measuring site-specific amide hydrogen-deuterium exchange rates for membrane proteins in bilayers is reported and evaluated. This method represents an adaptation and extension of the approach of Dempsey and co-workers (Biophys. J. 70, 1777-1788 (1996)) and is based on reconstituting 15N-labeled membrane proteins into phospholipid bilayers, followed by lyophilization and rehydration with D2O or H2O (control). Following incubation for a time t under hydrated conditions, samples are again lyophilized and then solubilized in an organic solvent system, where 1H-15N HSQC spectra are recorded. Comparison of spectra from D2O-exposed samples to spectra from control samples yields the extent of the H-D exchange which occurred in the bilayers during time t. Measurements are site specific if specific 15N labeling is used. The first part of this paper deals with the search for a suitable solvent system in which to solubilize complex membrane proteins in an amide "exchange-trapped" form for NMR quantitation of amide peak intensities. The second portion of the paper documents application of the overall procedure to measuring site-specific amide exchange rates in diacylglycerol kinase, a representative integral membrane protein. Both the potential usefulness and the significant limitations of the new method are documented.

  19. Automated Hydrogen/Deuterium Exchange Electron Transfer Dissociation High Resolution Mass Spectrometry Measured at Single-Amide Resolution

    Science.gov (United States)

    Landgraf, Rachelle R.; Chalmers, Michael J.; Griffin, Patrick R.

    2012-02-01

    Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique electron transfer dissociation (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing single-amide deuterium incorporation for differential HDX analysis of a nuclear receptor upon binding small molecule ligands. We are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established and fully automated HDX platform making differential single-amide HDX possible for bottom-up analysis of complex systems. We have applied this system to determine differential single amide resolution HDX data for the peroxizome proliferator activated receptor bound with two ligands of interest.

  20. Determination of Equine Cytochrome c Backbone Amide Hydrogen/Deuterium Exchange Rates by Mass Spectrometry Using a Wider Time Window and Isotope Envelope.

    Science.gov (United States)

    Hamuro, Yoshitomo

    2017-03-01

    A new strategy to analyze amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data is proposed, utilizing a wider time window and isotope envelope analysis of each peptide. While most current scientific reports present HDX-MS data as a set of time-dependent deuteration levels of peptides, the ideal HDX-MS data presentation is a complete set of backbone amide hydrogen exchange rates. The ideal data set can provide single amide resolution, coverage of all exchange events, and the open/close ratio of each amide hydrogen in EX2 mechanism. Toward this goal, a typical HDX-MS protocol was modified in two aspects: measurement of a wider time window in HDX-MS experiments and deconvolution of isotope envelope of each peptide. Measurement of a wider time window enabled the observation of deuterium incorporation of most backbone amide hydrogens. Analysis of the isotope envelope instead of centroid value provides the deuterium distribution instead of the sum of deuteration levels in each peptide. A one-step, global-fitting algorithm optimized exchange rate and deuterium retention during the analysis of each amide hydrogen by fitting the deuterated isotope envelopes at all time points of all peptides in a region. Application of this strategy to cytochrome c yielded 97 out of 100 amide hydrogen exchange rates. A set of exchange rates determined by this approach is more appropriate for a patent or regulatory filing of a biopharmaceutical than a set of peptide deuteration levels obtained by a typical protocol. A wider time window of this method also eliminates false negatives in protein-ligand binding site identification. Graphical Abstract ᅟ.

  1. Determination of Equine Cytochrome c Backbone Amide Hydrogen/Deuterium Exchange Rates by Mass Spectrometry Using a Wider Time Window and Isotope Envelope

    Science.gov (United States)

    Hamuro, Yoshitomo

    2017-03-01

    A new strategy to analyze amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data is proposed, utilizing a wider time window and isotope envelope analysis of each peptide. While most current scientific reports present HDX-MS data as a set of time-dependent deuteration levels of peptides, the ideal HDX-MS data presentation is a complete set of backbone amide hydrogen exchange rates. The ideal data set can provide single amide resolution, coverage of all exchange events, and the open/close ratio of each amide hydrogen in EX2 mechanism. Toward this goal, a typical HDX-MS protocol was modified in two aspects: measurement of a wider time window in HDX-MS experiments and deconvolution of isotope envelope of each peptide. Measurement of a wider time window enabled the observation of deuterium incorporation of most backbone amide hydrogens. Analysis of the isotope envelope instead of centroid value provides the deuterium distribution instead of the sum of deuteration levels in each peptide. A one-step, global-fitting algorithm optimized exchange rate and deuterium retention during the analysis of each amide hydrogen by fitting the deuterated isotope envelopes at all time points of all peptides in a region. Application of this strategy to cytochrome c yielded 97 out of 100 amide hydrogen exchange rates. A set of exchange rates determined by this approach is more appropriate for a patent or regulatory filing of a biopharmaceutical than a set of peptide deuteration levels obtained by a typical protocol. A wider time window of this method also eliminates false negatives in protein-ligand binding site identification.

  2. Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano-electrospray ionization mass spectrometry†

    Science.gov (United States)

    Guo, Mingquan; Huang, Bill X.; Kim, Hee-Yong

    2009-01-01

    Amide hydrogen exchange coupled to nano-electrospray ionization mass spectrometry (nano-ESI-MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D2O buffer, digested with pepsin, and analyzed by nano-ESI-MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation. In the PH domain, four β-strand (β1, β2 β5 and β6) regions containing membrane-binding residues displayed higher solvent accessibility in the inactive state, suggesting that the PH domain is readily available for the binding to the plasma membrane for activation. In contrast, these β-strands became less exposed or more folded in the active form, which is favored for the dissociation of Akt from the membrane. The beginning α-helix J region and the C-terminal locus (T450-470P) of the regulatory domain showed less folded structures that probably enable substrate entry. Our data also revealed detailed conformational changes of Akt in the kinase domain due to activation, some of which may be attributed to the interaction of the basic residues with phosphorylation sites. Our H/D exchange results indicating the conformational status of Akt at different activation states provided new insight for the regulation of this critical protein involved in cell survival. PMID:19462409

  3. Structural dynamics of soluble chloride intracellular channel protein CLIC1 examined by amide hydrogen-deuterium exchange mass spectrometry.

    Science.gov (United States)

    Stoychev, Stoyan H; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L; Dirr, Heini W

    2009-09-08

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1, but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2, and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilizing domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix alpha1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand beta2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer.

  4. Structural insights into glucan phosphatase dynamics using amide hydrogen-deuterium exchange mass spectrometry.

    Science.gov (United States)

    Hsu, Simon; Kim, Youngjun; Li, Sheng; Durrant, Eric S; Pace, Rachel M; Woods, Virgil L; Gentry, Matthew S

    2009-10-20

    Laforin and starch excess 4 (SEX4) are founding members of a class of phosphatases that dephosphorylate phosphoglucans. Each protein contains a carbohydrate binding module (CBM) and a dual-specificity phosphatase (DSP) domain. The gene encoding laforin is mutated in a fatal neurodegenerative disease called Lafora disease (LD). In the absence of laforin function, insoluble glucans that are hyperphosphorylated and exhibit sparse branching accumulate. It is hypothesized that these accumulations trigger the neurodegeneration and premature death of LD patients. We recently demonstrated that laforin removes phosphate from phosphoglucans and hypothesized that this function inhibits insoluble glucan accumulation. Loss of SEX4 function in plants yields a similar cellular phenotype; an excess amount of insoluble, hyperphosphorylated glucans accumulates in cells. While multiple groups have shown that these phosphatases dephosphorylate phosphoglucans, there is no structure of a glucan phosphatase and little is known about the mechanism whereby they perform this action. We utilized hydrogen-deuterium exchange mass spectrometry (DXMS) and structural modeling to probe the conformational and structural dynamics of the glucan phosphatase SEX4. We found that the enzyme does not undergo a global conformational change upon glucan binding but instead undergoes minimal rearrangement upon binding. The CBM has improved protection from deuteration when bound to glucans, confirming its role in glucan binding. More interestingly, we identified structural components of the DSP that also have improved protection from deuteration upon glucan addition. To determine the position of these regions, we generated a homology model of the SEX4 DSP. The homology model shows that all of these regions are adjacent to the DSP active site. Therefore, our results suggest that these regions of the DSP participate in the presentation of the phosphoglucan to the active site and provide the first structural

  5. Structural Insights of Glucan Phosphatase Dynamics using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Hsu, Simon; Kim, Youngjun; Li, Sheng; Durrant, Eric S.; Pace, Rachel M.; Woods, Virgil L.; Gentry, Matthew S.

    2009-01-01

    Laforin and Starch Excess 4 (SEX4) are founding members of a class of phosphatases that dephosphorylate phosphoglucans. Each protein contains a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. The gene encoding laforin is mutated in a fatal neurodegenerative disease called Lafora disease (LD). In the absence of laforin function, insoluble glucans accumulate that are hyperphosphorylated and exhibit sparse branching. It is hypothesized that these accumulations trigger the neurodegeneration and premature death of LD patients. We recently demonstrated that laforin removes phosphate from phosphoglucans and hypothesized that this function inhibits insoluble glucan accumulation. Loss of SEX4 function in plants yields a similar cellular phenotype; cells accumulate an excess amount of insoluble, hyperphosphorylated glucans. While multiple groups have shown that these phosphatases dephosphorylate phosphoglucans, there is no structure of a glucan phosphatase and little is known about the mechanism whereby they perform this action. We utilized hydrogen-deuterium exchange mass spectrometry (DXMS) and structural modeling to probe the conformational and structural dynamics of the glucan phosphatase SEX4. We found that the enzyme does not undergo a global conformational change upon glucan binding, but instead undergoes minimal rearrangement upon binding. The CBM undergoes increased protection from deuteration when bound to glucans, confirming its role in glucan binding. More interestingly, we identified structural components of the DSP that also undergo increased protection from deuteration upon glucan addition. To determine the position of these regions, we generated a homology model of the SEX4 DSP. The homology model shows that all of these regions are adjacent the DSP active site. Therefore, our results suggest that these regions of the DSP participate in presenting the phosphoglucan to the active site and provide the first structural analysis

  6. Dissecting interdomain communication within cAPK regulatory subunit type IIβ using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

    Science.gov (United States)

    Zawadzki, Kerri M.; Hamuro, Yoshitomo; Kim, Jack S.; Garrod, Siv; Stranz, David D.; Taylor, Susan S.; Woods, Virgil L.

    2003-01-01

    cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIβ subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain. PMID:12930997

  7. Hydrogen abstraction reactions by amide electron adducts

    International Nuclear Information System (INIS)

    Sevilla, M.D.; Sevilla, C.L.; Swarts, S.

    1982-01-01

    Electron reactions with a number of peptide model compounds (amides and N-acetylamino acids) in aqueous glasses at low temperature have been investigated using ESR spectroscopy. The radicals produced by electron attachment to amides, RC(OD)NDR', are found to act as hydrogen abstracting agents. For example, the propionamide electron adduct is found to abstract from its parent propionamide. Electron adducts of other amides investigated show similar behavior except for acetamide electron adduct which does not abstract from its parent compound, but does abstract from other amides. The tendency toward abstraction for amide electron adducts are compared to electron adducts of several carboxylic acids, ketones, aldehydes and esters. The comparison suggests the hydrogen abstraction tendency of the various deuterated electron adducts (DEAs) to be in the following order: aldehyde DEA > acid DEA = approximately ester DEA > ketone DEA > amide DEA. In basic glasses the hydrogen abstraction ability of the amide electron adducts is maintained until the concentration of base is increased sufficiently to convert the DEA to its anionic form, RC(O - )ND 2 . In this form the hydrogen abstracting ability of the radical is greatly diminished. Similar results were found for the ester and carboxylic acid DEA's tested. (author)

  8. Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS).

    Science.gov (United States)

    Moorthy, Balakrishnan S; Schultz, Steven G; Kim, Sherry G; Topp, Elizabeth M

    2014-06-02

    Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

  9. Structural elements involved in proton translocation by cytochrome c oxidase as revealed by backbone amide hydrogen-deuterium exchange of the E286H mutant.

    Science.gov (United States)

    Busenlehner, Laura S; Brändén, Gisela; Namslauer, Ida; Brzezinski, Peter; Armstrong, Richard N

    2008-01-08

    Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa3), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O2 reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains approximately 1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton translocation by CytcO.

  10. Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS).

    Science.gov (United States)

    Li, Sheng; Tsalkova, Tamara; White, Mark A; Mei, Fang C; Liu, Tong; Wang, Daphne; Woods, Virgil L; Cheng, Xiaodong

    2011-05-20

    Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Structural Dynamics of Soluble Chloride Intracellular Channel Protein CLIC1 Examined by Amide Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS)†

    Science.gov (United States)

    Stoychev, Stoyan H.; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L.; Dirr, Heini W.

    2009-01-01

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1 but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2 and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilising domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix α1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand β2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer. PMID:19650640

  12. Catalyst for hydrogen-amine D exchange

    International Nuclear Information System (INIS)

    Holtslander, W.J.; Johnson, R.E.

    1976-01-01

    A process is claimed for deuterium isotopic enrichment (suitable for use in heavy water production) by amine-hydrogen exchange in which the exchange catalyst comprises a mixture of alkyl amides of two metals selected from the group consisting of the alkali metals. Catalyst mixtures comprising at least one of the alkali amides of lithium and potassium are preferred. At least one of the following benefits are obtained: decreased hydride formation, decreased thermal decomposition of alkyl amide, increased catalyst solubility in the amine phase, and increased exchange efficiency. 11 claims

  13. Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation

    DEFF Research Database (Denmark)

    Jørgensen, Thomas J D; Gårdsvoll, Henrik; Ploug, Michael

    2005-01-01

    if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional...... are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity...... of this labeling was confirmed after pepsin proteolysis. CID of such deuterated peptides, [M + 2H](2+), yielded fragment ions (b- and y-ions) having a deuterium content that resemble the theoretical values calculated for 100% scrambling. Thus, complete randomization of all hydrogen atoms attached to nitrogen...

  14. On the determinants of amide backbone exchange in proteins: a neutron crystallographic comparative study.

    Science.gov (United States)

    Bennett, Brad C; Gardberg, Anna S; Blair, Matthew D; Dealwis, Chris G

    2008-07-01

    The hydrogen/deuterium-exchange (HDX) method, coupled with neutron diffraction, is a powerful probe for investigating molecular dynamics. In the present report, general determinants of HDX are proposed based on 12 deposited neutron protein structures. The parameters that correlate best with HDX are the depth within the protein structure of the amide N atom and the secondary-structure type. Both the B factor of the amide N atom and the ratio B/B correlate moderately. However, solvent accessibility only correlates strongly for one molecule and hydrogen-bonding distance correlates for two molecules with respect to amide HDX. In addition to the relatively small number of neutron structures available, the limitations to this type of analysis, namely resolution, data completeness and the data-to-parameter ratio, are discussed briefly. A global analysis of HDX was performed to overcome some of these obstacles, damping the effects of outliers and the extreme variation of the data sets arising from resolution limitations. From this, amide depth and hydrogen-bonding distance to the amide (a measure of interaction strength) show strong global correlation with HDX. For some structures, the constituents of the hydrophobic protein core could be identified based on contiguous regions that are resistant to exchange and have significant depth. These may, in fact, constitute minimal folding domains.

  15. Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange

    Science.gov (United States)

    Burns-Hamuro, Lora L.; Hamuro, Yoshitomo; Kim, Jack S.; Sigala, Paul; Fayos, Rosa; Stranz, David D.; Jennings, Patricia A.; Taylor, Susan S.; Woods, Virgil L.

    2005-01-01

    The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIα) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIα) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an α-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIα and RIIα D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIα compared to RIIα, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIα indicated that there were more binding constraints for the AKB ligand when bound to RIα. This was in contrast to RIIα, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIα AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein–ligand and protein–protein interactions. PMID:16260760

  16. Use of triphenyl phosphate as risk mitigant for metal amide hydrogen storage materials

    Energy Technology Data Exchange (ETDEWEB)

    Cortes-Concepcion, Jose A.; Anton, Donald L.

    2016-04-26

    A process in a resulting product of the process in which a hydrogen storage metal amide is modified by a ball milling process using an additive of TPP. The resulting product provides for a hydrogen storage metal amide having a coating that renders the hydrogen storage metal amide resistant to air, ambient moisture, and liquid water while improving useful hydrogen storage and release kinetics.

  17. Rapid and accurate processing method for amide proton exchange rate measurement in proteins

    International Nuclear Information System (INIS)

    Koskela, Harri; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami

    2007-01-01

    Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing

  18. Solvent Exchange Rates of Side-chain Amide Protons in Proteins

    International Nuclear Information System (INIS)

    Rajagopal, Ponni; Jones, Bryan E.; Klevit, Rachel E.

    1998-01-01

    Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr. The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons. Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8. An NOE between a side-chain amide proton and a bound water molecule was also observed

  19. Hydrogen isotope exchange reactions

    International Nuclear Information System (INIS)

    Jones, J.R.

    1980-01-01

    The two most widely used methods for following hydrogen isotope exchange reactions, namely dedeuteriation and detritiation, involve in the first place the synthesis of an appropriately labelled compound. Rates of dedeuteriation are usually followed by measuring changes in the 1 H n.m.r. spectrum of the substrate (examples are given); the method not only gives the rate but also the site(s) of exchange. It is limited to rather slow reactions and is not as accurate as some of the other methods. The development of deuterium n.m.r. spectroscopy means that changes in the 2 H n.m.r. spectrum can also be used to measure rates of dedeuteriation. The development of liquid scintillation counting greatly eased the problem of how to detect weak β emitters; the attractions of tritium as a tracer were thereby much enhanced. Nowadays the study of rates of detritiation constitutes one of the most versatile and accurate methods of following hydrogen isotope exchange. Examples of the technique are given. (U.K.)

  20. Catalysed hydrogen isotope exchange

    International Nuclear Information System (INIS)

    1973-01-01

    A method is described for enhancing the rate of exchange of hydrogen atoms in organic compounds or moieties with deuterium or tritium atoms. It comprises reacting the organic compound or moiety and a compound which is the source of deuterium or tritium in the presence of a catalyst consisting of a non-metallic, metallic or organometallic halide of Lewis acid character and which is reactive towards water, hydrogen halides or similar protonic acids. The catalyst is a halide or organometallic halide of: (i) zinc or another element of Group IIb; (ii) boron, aluminium or another element of Group III; (iii) tin, lead, antimony or another element of Groups IV to VI; or (iv) a transition metal, lanthanide or stable actinide; or a halohalide. (author)

  1. Electron capture dissociation proceeds with a low degree of intramolecular migration of peptide amide hydrogens

    DEFF Research Database (Denmark)

    Rand, Kasper D; Adams, Christopher M; Zubarev, Roman A

    2008-01-01

    ECD using peptides with a unique selective deuterium incorporation. Our results show that only limited amide hydrogen migration occurs upon ECD, provided that vibrational excitation prior to the electron capture event is minimized. Peptide ions that are excessively vibrationally excited......Hydrogen (1H/2H) exchange combined with mass spectrometry (HX-MS) has become a recognized method for the analysis of protein structural dynamics. Presently, the incorporated deuterons are typically localized by enzymatic cleavage of the labeled proteins and single residue resolution is normally...... only obtained for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation in tandem mass spectrometers would greatly increase the applicability of the HX-MS method. The biggest obstacle in achieving this goal is the intramolecular hydrogen migration (i.e., hydrogen...

  2. Hydrogen-bond detection in peptides by 1H-nuclear magnetic resonance through a hydrogen-chlorine exchange reaction

    International Nuclear Information System (INIS)

    Kondo, Michio; Nishi, Ichiro; Yamamoto, Makoto; Jelokhani-Niaraki, M.; Kodama, Hiroaki; Okamoto, Kouji.

    1994-01-01

    NMR spectroscopy is a versatile method for the conformational analysis of peptides and proteins. The hydrogen-chlorine exchange of amide NH protons is detected by 1 H NMR and used as a method to distinguish between intramolecularly hydrogen-bonded and solvent-exposed NH moieties. The method has been applied to hydrogen bond detection in naturally occurring antibiotic peptides, such as gramicidin S, and CH 3 CONH-X (X=alkyl- or aryl-) derivatives. The deuterium exchange method was compared with this method in parallel experiments. In the case of chlorine exchange, in contrast to deuterium exchange, the hydrogen-bonded amide protons are replaced much faster than their solvent-exposed counterparts and the duration of the experiments is considerably less. It is highly possible that the hydrogen-chlorine exchange reaction under the present experimental conditions, in the dark and at room temperature, proceeds through an electrophilic polar mechanism. (author)

  3. Regio-Selective Intramolecular Hydrogen/Deuterium Exchange in Gas-Phase Electron Transfer Dissociation

    Science.gov (United States)

    Hamuro, Yoshitomo

    2017-05-01

    Protein backbone amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) typically utilizes enzymatic digestion after the exchange reaction and before MS analysis to improve data resolution. Gas-phase fragmentation of a peptic fragment prior to MS analysis is a promising technique to further increase the resolution. The biggest technical challenge for this method is elimination of intramolecular hydrogen/deuterium exchange (scrambling) in the gas phase. The scrambling obscures the location of deuterium. Jørgensen's group pioneered a method to minimize the scrambling in gas-phase electron capture/transfer dissociation. Despite active investigation, the mechanism of hydrogen scrambling is not well-understood. The difficulty stems from the fact that the degree of hydrogen scrambling depends on instruments, various parameters of mass analysis, and peptide analyzed. In most hydrogen scrambling investigations, the hydrogen scrambling is measured by the percentage of scrambling in a whole molecule. This paper demonstrates that the degree of intramolecular hydrogen/deuterium exchange depends on the nature of exchangeable hydrogen sites. The deuterium on Tyr amide of neurotensin (9-13), Arg-Pro-Tyr-Ile-Leu, migrated significantly faster than that on Ile or Leu amides, indicating the loss of deuterium from the original sites is not mere randomization of hydrogen and deuterium but more site-specific phenomena. This more precise approach may help understand the mechanism of intramolecular hydrogen exchange and provide higher confidence for the parameter optimization to eliminate intramolecular hydrogen/deuterium exchange during gas-phase fragmentation.

  4. Hydrogen exchange and ligand binding: Ligand-dependent and ligand-independent protection in the Src SH3 domain

    OpenAIRE

    Wildes, David; Marqusee, Susan

    2005-01-01

    Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these high-energy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple...

  5. Measuring the hydrogen/deuterium exchange of proteins at high spatial resolution by mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper Dyrberg; Zehl, Martin; Jørgensen, Thomas J D

    2014-01-01

    the sum of the exchange kinetics for the individual backbone amides. Local exchange kinetics is typically achieved by using pepsin digestion under quench conditions (i.e., under cold acidic conditions where the amide hydrogen exchange rate is slowed by many orders of magnitude). The ability to localize...... resolution of the lowest-energy structure of the native ensemble. There is a growing need for sensitive analytical tools to explore all of the significant molecular structures in the conformational landscape of proteins. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has recently emerged...... as a powerful method for characterizing protein conformational dynamics. The basis of this method is the fact that backbone amides in stable hydrogen-bonded structures (e.g., α-helices and β-sheets) are protected against exchange with the aqueous solvent. All protein structures are dynamic, however...

  6. Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy.

    Science.gov (United States)

    del Amo, Juan-Miguel Lopez; Fink, Uwe; Reif, Bernd

    2010-12-01

    We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual (15)N-T (1) timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s(-1). Backbone amide (15)N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D(2)O is employed as a solvent for sample preparation. Due to the intrinsically long (15)N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

  7. Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    Lopez del Amo, Juan-Miguel; Fink, Uwe; Reif, Bernd

    2010-01-01

    We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual 15 N-T 1 timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s -1 . Backbone amide 15 N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D 2 O is employed as a solvent for sample preparation. Due to the intrinsically long 15 N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

  8. Catalyst for hydrogen-amine D exchange

    International Nuclear Information System (INIS)

    Holtslander, W.J.; Johnson, R.E.

    1977-01-01

    In a process for enrichment of deuterium by contacting hydrogen with an amine (such as methylamine), an alkali metal amide (such as potassium methylamide) is used as a catalyst. The present improvement is to use a mixture of two metal amides (e.g. lithium methylamide plus potassium methylamide) in order to prevent precipitation of a hydride and to reduce thermal decomposition of the catalyst. (NDH)

  9. Hydrogen and chlorine isotope exchange in hydrogen dichloride ions

    International Nuclear Information System (INIS)

    Szydlowski, J.; Ratajska, W.

    1987-01-01

    The kinetics of deuterium and chlorine-36 isotope exchange between hydrogen dichloride ions in tetramethyl- and tetraethyl-ammonium salts and hydrogen chloride was studied in the temperature range of 275-304 K. On the basis of the results obtained the exchange mechanism was proposed emphasizing the role of hydrogen bonding in the exchange process. (author)

  10. Gas-phase fragmentation of peptides by MALDI in-source decay with limited amide hydrogen (1H/2H) scrambling

    DEFF Research Database (Denmark)

    Bache, Nicolai; Rand, Kasper D; Roepstorff, Peter

    2008-01-01

    To achieve a fundamental understanding of the function of proteins and protein complexes at the molecular level, it is crucial to obtain a detailed knowledge about their dynamic and structural properties. The kinetics of backbone amide hydrogen exchange is intimately linked to the structural...... dynamics of the protein, and in recent years, the monitoring of the isotopic exchange of these hydrogens by mass spectrometry has become a recognized method. At present, the resolution of this method is, however, limited and single-residue resolution is typically only obtained for a few residues...... in a protein. It would therefore be desirable if gas-phase fragmentation could be used to localize incorporated deuterons as this would ultimately lead to single-residue resolution. A central obstacle for this approach is, however, the occurrence of intramolecular migration of amide hydrogens upon activation...

  11. Collisional activation by MALDI tandem time-of-flight mass spectrometry induces intramolecular migration of amide hydrogens in protonated peptides

    DEFF Research Database (Denmark)

    Jørgensen, Thomas J D; Bache, Nicolai; Roepstorff, Peter

    2005-01-01

    -specific information about the incorporation of deuterium into peptides and proteins in solution. Using a unique set of peptides with their carboxyl-terminal half labeled with deuterium we have shown unambiguously that hydrogen (1H/2H) scrambling is such a dominating factor during low energy collisional activation...... of doubly protonated peptides that the original regioselective deuterium pattern of these peptides is completely erased (Jørgensen, T. J. D., Gårdsvoll, H., Ploug, M., and Roepstorff, P. (2005) Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation. J. Am. Chem. Soc...... randomization among all exchangeable sites (i.e. all N- and O-linked hydrogens) also occurs upon high energy collisional activation of singly protonated peptides. This intense proton/deuteron traffic precludes the use of MALDI tandem time-of-flight mass spectrometry to obtain reliable information...

  12. Prediction of amino acid residues protected from hydrogen-deuterium exchange in a protein chain.

    Science.gov (United States)

    Dovidchenko, N V; Lobanov, M Yu; Garbuzynskiy, S O; Galzitskaya, O V

    2009-08-01

    We have investigated the possibility to predict protection of amino acid residues from hydrogen-deuterium exchange. A database containing experimental hydrogen-deuterium exchange data for 14 proteins for which these data are known has been compiled. Different structural parameters related to flexibility of amino acid residues and their amide groups have been analyzed to answer the question whether these parameters can be used for predicting the protection of amino acid residues from hydrogen-deuterium exchange. A method for prediction of protection of amino acid residues, which uses only the amino acid sequence of a protein, has been elaborated.

  13. [Interactions between proteins and cation exchange adsorbents analyzed by NMR and hydrogen/deuterium exchange technique].

    Science.gov (United States)

    Wang, Kang; Hao, Dongxia; Qi, Shuting; Ma, Guanghui

    2014-09-01

    In silico acquirement of the accurate residue details of protein on chromatographic media is a bottleneck in protein chromatography separation and purification. Here we developed a novel approach by coupling with H/D exchange and nuclear magnetic resonance to observe hen egg white lysozyme (HEWL) unfolding behavior adsorbed on cation exchange media (SP Sepharose FF). Analysis of 1D 1H-NMR shows that protein unfolding accelerated H/D exchange rate, leading to more loss of signal of amide hydrogen owing to exposure of residues and the more unfolding of protein. Analysis of two-dimensional hydrogen-hydrogen total correlation spectroscopy shows that lysozyme lost more signals and experienced great unfolding during its adsorption on media surface. However, for several distinct fragments, the protection degrees varied, the adsorbed lysozyme lost more signal intensity and was less protected at disorder structures (coil, bend, and turn), but was comparatively more protected against exchange at secondary structure domains (α-helix, β-sheet). Finally, the binding site was determined by electrostatic calculations using computer simulation methods in conjunction with hydrogen deuterium labeled protein and NMR. This study would help deeply understand the microscopic mechanism of protein chromatography and guide the purposely design of chromatographic process and media. Moreover, it also provide an effective tool to study the protein and biomaterials interaction in other applications.

  14. Hydrogen storage by reaction between metallic amides and imides

    International Nuclear Information System (INIS)

    Eymery, J.B.; Cahen, S.; Tarascon, J.M.; Janot, R.

    2007-01-01

    This paper details the various metal-N-H systems reported in the literature as possible hydrogen storage materials. In a first part, we discuss the hydrogen storage performances of the Li-N-H system and the desorption mechanism of the LiH-LiNH 2 mixture is especially presented. The possibility of storing hydrogen using two other binary systems (Mg-N-H and Ca-N-H) is described in a second part. In the third part of the paper, we discuss about the performances of the highly promising Li-Mg-N-H system, for which a nice reversibility is obtained at 200 C with an experimental hydrogen capacity of about 5.0 wt.%. Other ternary systems, as Li-B-N-H and Li-Al-N-H, are presented in the last part of this review paper. We especially emphasize the performances obtained in our Laboratory at Amiens with a LiAl(NH 2 ) 4 -LiH mixture able to desorb around 6.0 wt.% of hydrogen at only 130 C. (authors)

  15. Effect of molecular weight on hydrogen--deuterium exchange in a nonhelical polyamide

    International Nuclear Information System (INIS)

    Snyder, W.D.; Klotz, I.M.

    1975-01-01

    Hydrogen--deuterium exchange kinetics were measured for poly(isopropylacrylamides) over a molecular weight range from 100 to 200,000. A sharp drop in rate appears as soon as one goes from monomer to trimer. Thereafter a very slow progressive decrease is displayed. The local environment of the exchanging amide residue is largely (although not completely) established by the mutual interaction of as few as three residues in these polymers of random conformation. (U.S.)

  16. Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

    Science.gov (United States)

    Englander, Joan J.; Del Mar, Charyl; Li, Will; Englander, S. Walter; Kim, Jack S.; Stranz, David D.; Hamuro, Yoshitomo; Woods, Virgil L.

    2003-01-01

    An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy. PMID:12773622

  17. Mapping Protein-Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry.

    Science.gov (United States)

    Gallagher, Elyssia S; Hudgens, Jeffrey W

    2016-01-01

    Biological processes are the result of noncovalent, protein-ligand interactions, where the ligands range from small organic and inorganic molecules to lipids, nucleic acids, peptides, and proteins. Amide groups within proteins constantly exchange protons with water. When immersed in heavy water (D2O), mass spectrometry (MS) can measure the change of mass associated with the hydrogen to deuterium exchange (HDX). Protein-ligand interactions modify the hydrogen exchange rates of amide protons, and the measurement of the amide exchange rates can provide rich information regarding the dynamical structure of the protein-ligand complex. This chapter describes a protocol for conducting bottom-up, continuous uptake, proteolytic fragmentation HDX-MS experiments that can help identify and map the interacting peptides of a protein-ligand interface. This tutorial outlines the fundamental theory governing hydrogen exchange; provides practical information regarding the preparation of protein samples and solutions; and describes the exchange reaction, reaction quenching, enzymatic digestion, chromatographic separation, and peptide analysis by MS. Tables list representative combinations of fluidic components used by HDX-MS researchers and summarize the available HDX-MS analysis software packages. Additionally, two HDX-MS case studies are used to illustrate protein-ligand interactions involving: (1) a continuous sequence of interacting residues and (2) a set of discontinuously numbered residues, residing spatially near each other. © 2016 Elsevier Inc. All rights reserved.

  18. Probing the role of backbone hydrogen bonds in protein-peptide interactions by amide-to-ester mutations

    DEFF Research Database (Denmark)

    Eildal, Jonas N N; Hultqvist, Greta; Balle, Thomas

    2013-01-01

    of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding...

  19. Kinetic Modification on Hydrogen Desorption of Lithium Hydride and Magnesium Amide System

    OpenAIRE

    Miyaoka, Hiroki; Wang, Yongming; Hino, Satoshi; Isobe, Shigehito; Tokoyoda, Kazuhiko; Ichikawa, Takayuki; Kojima, Yoshitsugu

    2015-01-01

    Various synthesis and rehydrogenation processes of lithium hydride (LiH) and magnesium amide (Mg(NH2)2) system with 8:3 molar ratio are investigated to understand the kinetic factors and effectively utilize the essential hydrogen desorption properties. For the hydrogen desorption with a solid-solid reaction, it is expected that the kinetic properties become worse by the sintering and phase separation. In fact, it is experimentally found that the low crystalline size and the close contact of L...

  20. The hydrogen-deuterium exchange of macromolecules

    International Nuclear Information System (INIS)

    Duane, C.J.

    1982-06-01

    An infra red spectroscopic technique has been developed which allows the hydrogen-deuterium exchange between a polymer and absorbed heavy water to be monitored with the polymer in contact with liquid heavy water. This method extends the existing methods to the highest possible internal water concentration. The results of Moore on the hydrogen-deuterium exchange of poly(N-t-butylacrylamide) have been extended to the lower homologues of the series. The rate and extent of the exchange of these polymers is dependent upon the pH of the conditioning solution in which the polymer is exposed prior to the exchange study. The exchange is acid and base catalysed with the latter only apparent in the lower homologues. The features of the acid catalysed exchange have been tentatively explained in terms of the mobility of the hydrogen ion in the polymer matrix, whilst the restricted base exchange is attributed to the lack of hydration stabilisation of the intermediate ion complex of the base catalysed mechanism. The hydrogen-deuterium exchange properties of nylon, silk and cellulose have also been studied and these polymers show far less sensitivity to the pH of the conditioning treatment. The results are discussed. (author)

  1. The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

    Directory of Open Access Journals (Sweden)

    Tsinoremas NF

    2007-05-01

    Full Text Available Abstract Background The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues. Results We have developed an integrated software system for the automated analysis and representation of H/D exchange data that has been titled "The Deuterator". Novel approaches have been implemented that enable high throughput analysis, automated determination of deuterium incorporation, and deconvolution of overlapping peptides. This has been achieved by using methods involving iterative theoretical envelope fitting, and consideration of peak data within expected m/z ranges. Existing common file formats have been leveraged to allow compatibility with the output from the myriad of MS instrument platforms and peptide sequence database search engines. A web-based interface is used to integrate the components of The Deuterator that are able to analyze and present mass spectral data from instruments with varying resolving powers. The results, if necessary, can then be confirmed, adjusted, re-calculated and saved. Additional tools synchronize the curated calculation parameters with replicate time points, increasing throughput. Saved results can then

  2. Deuterium-hydrogen monothermal exchange

    International Nuclear Information System (INIS)

    Rae, H.K.

    1975-01-01

    A monothermal exchange process of extracting deuterium from ammonia synthesis gas is described. This process comprises passing the gas through an exchage liquid stream consisting of a liquid amine having up to five carbon atoms per molecule to cause deuterium to be transferred from the synthesis gas to the exchange liquid, and removing a stream of exchange liquid enriched in deuterium therefrom. (Patent Office Record)

  3. Analytical aspects of hydrogen exchange mass spectrometry

    Science.gov (United States)

    Engen, John R.; Wales, Thomas E.

    2016-01-01

    The analytical aspects of measuring hydrogen exchange by mass spectrometry are reviewed. The nature of analytical selectivity in hydrogen exchange is described followed by review of the analytical tools required to accomplish fragmentation, separation, and the mass spectrometry measurements under restrictive exchange quench conditions. In contrast to analytical quantitation that relies on measurements of peak intensity or area, quantitation in hydrogen exchange mass spectrometry depends on measuring a mass change with respect to an undeuterated or deuterated control, resulting in a value between zero and the maximum amount of deuterium that could be incorporated. Reliable quantitation is a function of experimental fidelity and to achieve high measurement reproducibility, a large number of experimental variables must be controlled during sample preparation and analysis. The method also reports on important qualitative aspects of the sample, including conformational heterogeneity and population dynamics. PMID:26048552

  4. Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry

    DEFF Research Database (Denmark)

    Mysling, Simon; Salbo, Rune; Ploug, Michael

    2014-01-01

    requires a high concentration (>200 mM) of the chemical reducing agent Tris(2-carboxyethyl)phosphine (TCEP) as the reduction rate constant is decreased at low pH and temperature. Serious adverse effects on chromatographic and mass spectrometric performances have been reported when using high concentrations......Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0°C). The reduction typically...

  5. Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

    International Nuclear Information System (INIS)

    Downer, N.W.; Bruchman, T.J.; Hazzard, J.H.

    1986-01-01

    Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins

  6. Equilibrium studies on liquid ammonia-potassium amide-hydrogen system at high pressures and temperatures (Preprint No. CA-10)

    International Nuclear Information System (INIS)

    Donde, M.M.; Srinivasa, K.; Raman, S.

    1989-04-01

    Insoluble deposits were observed to form during the operation of Heavy Water Plant at Talcher, based on ammonia-hydrogen exchange process with potassium amide as catalyst. Experiments were undertaken to investigate this phenomenon under controlled conditions in the laboratory employing high pressures and elevated temperatures. It was observed that deposit formation was very minimal in the autoclave and no visible deposit was left on the strainer-filter, in all the experiments. Deposit analysis showed the presence of potassium hydroxide monohydrate as major component and alpha-potassium hydroxide and potassium azide as minor components. It is suggested that the presence of hydroxide may be due to the reaction of amide with the residual moisture in the system during the experiment and any ingress of moisture while opening for collection of deposit. Azide formation is explained by following reactions occurring during the experiments. NH 3 +KNH 2 →NH 2 -NH 2 +KH; NH 2 -NH 2 +KNH 2 →KN 3 +3H 2 . (author). 1 fig

  7. Guanidinium chloride induction of partial unfolding in amide proton exchange in RNase A.

    Science.gov (United States)

    Mayo, S L; Baldwin, R L

    1993-11-05

    Amide (NH) proton exchange rates were measured in 0.0 to 0.7 M guanidinium chloride (GdmCl) for 23 slowly exchanging peptide NH protons of ribonuclease A (RNase A) at pH* 5.5 (uncorrected pH measured in D2O), 34 degrees C. The purpose was to find out whether GdmCl induces exchange through binding to exchange intermediates that are partly or wholly unfolded. It was predicted that, when the logarithm of the exchange rate is plotted as a function of the molarity of GdmCl, the slope should be a measure of the amount of buried surface area exposed to GdmCl in the exchange intermediate. The results indicate that these concentrations of GdmCl do induce exchange by means of a partial unfolding mechanism for all 23 protons; this implies that exchange reactions can be used to study the unfolding and stability of local regions. Of the 23 protons, nine also show a second mechanism of exchange at lower concentrations of GdmCl, a mechanism that is nearly independent of GdmCl concentration and is termed "limited structural fluctuation."

  8. Hydrogen/deuterium exchange in mass spectrometry.

    Science.gov (United States)

    Kostyukevich, Yury; Acter, Thamina; Zherebker, Alexander; Ahmed, Arif; Kim, Sunghwan; Nikolaev, Eugene

    2018-03-30

    The isotopic exchange approach is in use since the first observation of such reactions in 1933 by Lewis. This approach allows the investigation of the pathways of chemical and biochemical reactions, determination of structure, composition, and conformation of molecules. Mass spectrometry has now become one of the most important analytical tools for the monitoring of the isotopic exchange reactions. Investigation of conformational dynamics of proteins, quantitative measurements, obtaining chemical, and structural information about individual compounds of the complex natural mixtures are mainly based on the use of isotope exchange in combination with high resolution mass spectrometry. The most important reaction is the Hydrogen/Deuterium exchange, which is mainly performed in the solution. Recently we have developed the approach allowing performing of the Hydrogen/Deuterium reaction on-line directly in the ionization source under atmospheric pressure. Such approach simplifies the sample preparation and can accelerate the exchange reaction so that certain hydrogens that are considered as non-labile will also participate in the exchange. The use of in-ionization source H/D exchange in modern mass spectrometry for structural elucidation of molecules serves as the basic theme in this review. We will focus on the mechanisms of the isotopic exchange reactions and on the application of in-ESI, in-APCI, and in-APPI source Hydrogen/Deuterium exchange for the investigation of petroleum, natural organic matter, oligosaccharides, and proteins including protein-protein complexes. The simple scenario for adaptation of H/D exchange reactions into mass spectrometric method is also highlighted along with a couple of examples collected from previous studies. © 2018 Wiley Periodicals, Inc.

  9. Kinetic Modification on Hydrogen Desorption of Lithium Hydride and Magnesium Amide System

    Directory of Open Access Journals (Sweden)

    Hiroki Miyaoka

    2015-06-01

    Full Text Available Various synthesis and rehydrogenation processes of lithium hydride (LiH and magnesium amide (Mg(NH22 system with 8:3 molar ratio are investigated to understand the kinetic factors and effectively utilize the essential hydrogen desorption properties. For the hydrogen desorption with a solid-solid reaction, it is expected that the kinetic properties become worse by the sintering and phase separation. In fact, it is experimentally found that the low crystalline size and the close contact of LiH and Mg(NH22 lead to the fast hydrogen desorption. To preserve the potential hydrogen desorption properties, thermochemical and mechanochemical rehydrogenation processes are investigated. Although the only thermochemical process results in slowing the reaction rate due to the crystallization, the ball-milling can recover the original hydrogen desorption properties. Furthermore, the mechanochemical process at 150 °C is useful as the rehydrogenation technique to preserve the suitable crystalline size and mixing state of the reactants. As a result, it is demonstrated that the 8LiH and 3Mg(NH22 system is recognized as the potential hydrogen storage material to desorb more than 5.5 mass% of H2 at 150 °C.

  10. 2D IR cross peaks reveal hydrogen-deuterium exchange with single residue specificity.

    Science.gov (United States)

    Dunkelberger, Emily B; Woys, Ann Marie; Zanni, Martin T

    2013-12-12

    A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

  11. Local disorder in hydrogen storage compounds: the case of lithium amide/imide.

    Science.gov (United States)

    Ludueña, Guillermo A; Wegner, Martin; Bjålie, Lars; Sebastiani, Daniel

    2010-08-02

    Amides and imides of alkali metals are a very promising class of materials for use as a hydrogen-storage system, as they are able to store and release hydrogen via a chemical route at controllable temperatures and pressures. We critically revise the present picture of the atomic structure of the lightest member (LiNH(2)/Li(2)NH) by using a combined computational and experimental approach. Specifically, ab initio path integral molecular dynamics simulations and solid-state (1)H NMR techniques are combined. The results show that the presently assumed local structure might be inconsistent or at least incomplete and needs considerable revision. In particular, the Li atoms turn out to be more mobile and more disordered than suggested by structural data obtained from X-ray scattering. Also, the configuration of the hydrogen atoms, which is accessible via the NMR experiment and the corresponding first-principles calculations, is different from the previously assumed data. The computed and experimentally observed (1)H NMR parameters are in very good mutual agreement and illustrate the unusual chemical environment of the hydrogen atoms in this system. Incorporating our results on the new lithium data, we show that the effect of nuclear quantum delocalization for the hydrogen atoms is considerably reduced compared to the perfect crystal structure.

  12. Unusual intramolecular CHO hydrogen bonding interaction between a sterically bulky amide and uranyl oxygen.

    Science.gov (United States)

    Kannan, Shanmugaperumal; Kumar, Mukesh; Sadhu, Biswajit; Jaccob, Madhavan; Sundararajan, Mahesh

    2017-12-12

    The selective separation of toxic heavy metals such as uranyl can be accomplished using ligands with stereognostic hydrogen bonding interactions to the uranyl oxo group, as proposed by Raymond and co-workers (T. S. Franczyk, K. R. Czerwinski and K. N. Raymond, J. Am. Chem. Soc., 1992, 114, 8138-8146). Recently, several ligands possessing this weak interaction have been proposed involving the hydrogen bonding of NH and OH based moieties with uranyl oxygen. We herein report the structurally and spectroscopically characterized CHO hydrogen bonding using a sterically bulky amide based ligand. In conjunction with experiments, electronic structure calculations are carried out to understand the structure, binding and the strength of the CHO hydrogen bonding interactions. This weak interaction is mainly due to the steric effect caused by a bulky substituent around the donor group which has direct relevance in designing novel ligands in nuclear waste management processes. Although the kinetics are very slow, the ligand is also highly selective to uranyl in the presence of other interfering ions such as lanthanides.

  13. Probing protein ensemble rigidity and hydrogen-deuterium exchange.

    Science.gov (United States)

    Sljoka, Adnan; Wilson, Derek

    2013-10-01

    Protein rigidity and flexibility can be analyzed accurately and efficiently using the program floppy inclusion and rigid substructure topography (FIRST). Previous studies using FIRST were designed to analyze the rigidity and flexibility of proteins using a single static (snapshot) structure. It is however well known that proteins can undergo spontaneous sub-molecular unfolding and refolding, or conformational dynamics, even under conditions that strongly favor a well-defined native structure. These (local) unfolding events result in a large number of conformers that differ from each other very slightly. In this context, proteins are better represented as a thermodynamic ensemble of 'native-like' structures, and not just as a single static low-energy structure. Working with this notion, we introduce a novel FIRST-based approach for predicting rigidity/flexibility of the protein ensemble by (i) averaging the hydrogen bonding strengths from the entire ensemble and (ii) by refining the mathematical model of hydrogen bonds. Furthermore, we combine our FIRST-ensemble rigidity predictions with the ensemble solvent accessibility data of the backbone amides and propose a novel computational method which uses both rigidity and solvent accessibility for predicting hydrogen-deuterium exchange (HDX). To validate our predictions, we report a novel site specific HDX experiment which characterizes the native structural ensemble of Acylphosphatase from hyperthermophile Sulfolobus solfataricus (Sso AcP). The sub-structural conformational dynamics that is observed by HDX data, is closely matched with the FIRST-ensemble rigidity predictions, which could not be attained using the traditional single 'snapshot' rigidity analysis. Moreover, the computational predictions of regions that are protected from HDX and those that undergo exchange are in very good agreement with the experimental HDX profile of Sso AcP.

  14. Probing protein ensemble rigidity and hydrogen-deuterium exchange

    Science.gov (United States)

    Sljoka, Adnan; Wilson, Derek

    2013-10-01

    Protein rigidity and flexibility can be analyzed accurately and efficiently using the program floppy inclusion and rigid substructure topography (FIRST). Previous studies using FIRST were designed to analyze the rigidity and flexibility of proteins using a single static (snapshot) structure. It is however well known that proteins can undergo spontaneous sub-molecular unfolding and refolding, or conformational dynamics, even under conditions that strongly favor a well-defined native structure. These (local) unfolding events result in a large number of conformers that differ from each other very slightly. In this context, proteins are better represented as a thermodynamic ensemble of ‘native-like’ structures, and not just as a single static low-energy structure. Working with this notion, we introduce a novel FIRST-based approach for predicting rigidity/flexibility of the protein ensemble by (i) averaging the hydrogen bonding strengths from the entire ensemble and (ii) by refining the mathematical model of hydrogen bonds. Furthermore, we combine our FIRST-ensemble rigidity predictions with the ensemble solvent accessibility data of the backbone amides and propose a novel computational method which uses both rigidity and solvent accessibility for predicting hydrogen-deuterium exchange (HDX). To validate our predictions, we report a novel site specific HDX experiment which characterizes the native structural ensemble of Acylphosphatase from hyperthermophile Sulfolobus solfataricus (Sso AcP). The sub-structural conformational dynamics that is observed by HDX data, is closely matched with the FIRST-ensemble rigidity predictions, which could not be attained using the traditional single ‘snapshot’ rigidity analysis. Moreover, the computational predictions of regions that are protected from HDX and those that undergo exchange are in very good agreement with the experimental HDX profile of Sso AcP.

  15. Infrared spectroscopy of the amide I mode of N-methylacetamide in solid hydrogen at 2-4 K.

    Science.gov (United States)

    Paulson, Leif O; Anderson, David T

    2011-11-24

    We report high-resolution (0.05 cm(-1)) FTIR spectra of the fundamental and first overtone of the amide I mode of trans-N-methylacetamide (NMA) trapped in solid molecular hydrogen (SMH) at cryogenic temperatures with low (0.03%) and high (55%) ortho-hydrogen (oH(2)) concentrations. NMA-doped SMH samples with high oH(2) concentrations are nearly free from inhomogeneous broadening, permitting the measured amide I homogeneous line width of 1.268(8) cm(-1) to be used to place a lower limit on the vibrational lifetime of 4.19(3) ps. Direct observation of the amide I overtone allows the harmonic vibrational frequency ω(e) = 1726.6(5) cm(-1) and the anharmonicity constant ω(e)x(e) = 8.5(2) cm(-1) to be determined for NMA isolated in SMH samples with low oH(2) concentrations. © 2011 American Chemical Society

  16. Simple Synthesis Hydrogenated Castor Oil Fatty Amide Wax and Its Coating Characterization.

    Science.gov (United States)

    Yu, Xiuzhu; Wang, Ning; Zhang, Rui; Zhao, Zhong

    2017-07-01

    A simple method for incorporating amine groups in hydrogenated castor oil (HCO) to produce wax for beeswax or carnauba wax substitution in packaging and coating was developed. From the conversion rate of the products, HCO was reacted with ethanolamine at 150°C for 5 h, and the molar ratio of HCO and ethanolamine was 1:4. The hardness of the final product was seven times higher than that of beeswax, the cohesiveness of the final product was 1.3 times higher than that of beeswax and approximately one half of that of carnauba wax, and the melting point of the final product is 98°C. The Fourier transform Infrared spectroscopy showed that the amide groups were incorporated to form the amide products. In coating application, the results showed that the force of the final product coating cardboard was higher than that of beeswax and paraffin wax and less than that of carnauba wax. After 24 h soaking, the compression forces were decreased. HCO fatty acid wax can be an alternative wax for carnauba wax and beeswax in coating applications.

  17. Hydrogen-water isotopic exchange process

    International Nuclear Information System (INIS)

    Cheung, H.

    1984-01-01

    The objects of this invention are achieved by a dual temperature isotopic exchange process employing hydrogen-water exchange with water passing in a closed recirculation loop between a catalyst-containing cold tower and the upper portion of a catalyst-containing hot tower, with feed water being introduced to the lower portion of the hot tower and being maintained out of contact with the water recirculating in the closed loop. Undue retarding of catalyst activity during deuterium concentration can thus be avoided. The cold tower and the upper portion of the hot tower can be operated with relatively expensive catalyst material of higher catalyst activity, while the lower portion of the hot tower can be operated with a relatively less expensive, more rugged catalyst material of lesser catalyst activity. The feed water stream, being restricted solely to the lower portion of the hot tower, requires minimal pretreatment for the removal of potential catalyst contaminants. The catalyst materials are desirably coated with a hydrophobic treating material so as to be substantially inaccessible to liquid water, thereby retarding catalyst fouling while being accessible to the gas for enhancing isotopic exchange between hydrogen gas and water vapor. A portion of the water of the closed loop can be passed to a humidification zone to heat and humidify the circulating hydrogen gas and then returned to the closed loop

  18. Localized hydration in lyophilized myoglobin by hydrogen-deuterium exchange mass spectrometry. 1. Exchange mapping.

    Science.gov (United States)

    Sophocleous, Andreas M; Zhang, Jun; Topp, Elizabeth M

    2012-04-02

    The local effects of hydration on myoglobin (Mb) in solid matrices containing mannitol or sucrose (1:1 w/w, protein:additive) were mapped using hydrogen-deuterium exchange with mass spectrometric analysis (HDX-MS) at 5 °C and compared to solution controls. Solid powders were exposed to D₂O(g) at controlled activity (a(w)) followed by reconstitution and analysis of the intact protein and peptides produced by pepsin digestion. HDX varied with matrix type, a(w), and position along the protein backbone. HDX was less in sucrose matrices than in mannitol matrices at all a(w) while the difference in solution was negligible. Differences in HDX in the two matrices were detectable despite similarities in their bulk water content. The extent of exchange in solids is proposed as a measure of the hydration of exchangeable amide groups, as well as protein conformation and dynamics; pepsin digestion allows these effects to be mapped with peptide-level resolution.

  19. Probing acid-amide intermolecular hydrogen bonding by NMR spectroscopy and DFT calculations

    Science.gov (United States)

    Chaudhari, Sachin Rama; Suryaprakash, N.

    2012-05-01

    Benzene carboxylic acids and benzamide act as their self-complement in molecular recognition to form inter-molecular hydrogen bonded dimers between amide and carboxylic acid groups, which have been investigated by 1H, 13C and 15N NMR spectroscopy. Extensive NMR studies using diffusion ordered spectroscopy (DOSY), variable temperature 1D, 2D NMR, established the formation of heterodimers of benzamide with benzoic acid, salicylic acid and phenyl acetic acid in deuterated chloroform solution. Association constants for the complex formation in the solution state have been determined. The results are ascertained by X-ray diffraction in the solid state. Intermolecular interactions in solution and in solid state were found to be similar. The structural parameters obtained by X-ray diffraction studies are compared with those obtained by DFT calculations.

  20. Spatially resolved protein hydrogen exchange measured by matrix-assisted laser desorption ionization in-source decay

    DEFF Research Database (Denmark)

    Rand, Kasper D; Bache, Nicolai; Nedertoft, Morten M

    2011-01-01

    Mass spectrometry has become a powerful tool for measuring protein hydrogen exchange and thereby reveal the structural dynamics of proteins in solution. Here we describe the successful application of a matrix-assisted laser desorption ionization (MALDI) mass spectrometry approach based on in......-source decay (ISD) to measure spatially resolved amide backbone hydrogen exchange. By irradiating deuterated protein molecules in a crystalline matrix with a high laser fluence, they undergo prompt fragmentation. Spatially resolved deuteration levels are readily obtained by mass analysis of consecutive...... fragment ions. MALDI ISD analysis of deuterated cytochrome c yielded an extensive series of c-fragment ions which originate from cleavage of nearly all N-C(α) bonds (Cys17 to Glu104) allowing for a detailed analysis of the deuterium content of the backbone amides. While hydrogen scrambling can be major...

  1. HDX Match Software for the Data Analysis of Top-Down ECD-FTMS Hydrogen/Deuterium Exchange Experiments

    Science.gov (United States)

    Petrotchenko, Evgeniy V.; Borchers, Christoph H.

    2015-11-01

    Hydrogen/deuterium exchange (HDX) combined with mass spectrometry is a powerful technique for studying protein structure. The recently developed top-down ECD-FTMS HDX approach (Pan J. et al., JACS, 2008) allows determination of the hydrogen/deuterium exchange of a protein's amide bonds, down to the single residue resolution. One of the existing limitations of this technology has been the laborious manual analysis of the MS/MS spectra. Here we present a software program for processing the data from these experiments. This program assigns the c- and z-fragment ion series of the protein, and calculates the number of the exchanged amide protons for each fragment by fitting the theoretically predicted isotopic envelopes of the deuterated fragments to the experimental data.

  2. Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.

    Science.gov (United States)

    Grohe, Kristof; Movellan, Kumar Tekwani; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Linser, Rasmus

    2017-05-01

    We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1 H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

  3. Localized hydration in lyophilized myoglobin by hydrogen-deuterium exchange mass spectrometry. 2. Exchange kinetics.

    Science.gov (United States)

    Sophocleous, Andreas M; Topp, Elizabeth M

    2012-04-02

    Solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX) is a promising method for characterizing proteins in amorphous solids. Though analysis of HDX kinetics is informative and well-established in solution, application of these methods to solid samples is complicated by possible heterogeneities in the solid. The studies reported here provide a detailed analysis of the kinetics of hydration and ssHDX for equine myoglobin (Mb) in solid matrices containing sucrose or mannitol. Water sorption was rapid relative to ssHDX, indicating that ssHDX kinetics was not limited by bulk water transport. Deuterium uptake in solids was well-characterized by a biexponential model; values for regression parameters provided insight into differences between the two solid matrices. Analysis of the widths of peptide mass envelopes revealed that, in solution, an apparent EX2 mechanism prevails, consistent with native conformation of the protein. In contrast, in mannitol-containing samples, a smaller non-native subpopulation exchanges by an EX1-like mechanism. Together, the results indicate that the analysis of ssHDX kinetic data and of the widths of peptide mass envelopes is useful in screening solid formulations of protein drugs for the presence of non-native species that cannot be detected by amide I FTIR.

  4. Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper D; Zehl, Martin; Jensen, Ole Nørregaard

    2009-01-01

    Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin...... proteolysis of the labeled protein, the assignment of deuteriums to individual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure...... the HX of individual amide linkages in the amyloidogenic protein beta2-microglobulin. A comparison of the deuterium levels of 60 individual backbone amides of beta2-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation...

  5. Fast hydrogen exchange affects 15N relaxation measurements in intrinsically disordered proteins

    International Nuclear Information System (INIS)

    Kim, Seho; Wu, Kuen-Phon; Baum, Jean

    2013-01-01

    Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, 1 H– 15 N HSQC, is used to measure the 15 N transverse relaxation rate (R 2 ), the measured R 2 rate is convoluted with the HX rate (k HX ) and has higher apparent R 2 values. Since the 15 N R 2 measurement is important for analyzing protein backbone dynamics, the HX effect on the R 2 measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing 15 N R 2 CPMG experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R 2 CPMG can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D 2 O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R 2 CPMG values be obtained by methods described herein.

  6. Process for exchanging tritium between gaseous hydrogen and water

    International Nuclear Information System (INIS)

    Hindin, S.G.; Roberts, G.W.

    1981-01-01

    An improved method of exchanging and concentrating the radioactive isotope of hydrogen from water or hydrogen gas is described. This heavy water enrichment system involves a low pressure, dual temperature process. (U.K.)

  7. Isotopic hydrogen exchange - a general method for the preparation of labelled organic compounds

    International Nuclear Information System (INIS)

    Zatsepina, N.N.; Tupitsyn, I.F.

    1977-01-01

    Presented are results of the development of an efficient method for tritium (deuterium) labelling of various organic compounds, based on isotopic hydrogen exchange reaction. The factors which determine the exchange reaction rate were studied in selected aliphatic, aromatic and heterocyclic compounds and in methyl derivatives of organic compounds containing nonmetallic elements. Solutions of sodium (potassium) hydroxide in water, sodium (potassium) alcoholate in alcohol and sodium (potassium) amide in liquid ammonia were used as solvents-donors of the heavy hydrogen isotope. The results of the kinetic studies were used for the choice of conditions for the preparation of a number of labelled, biologically important substances, solvents, polymer materials and compounds for general chemical use

  8. Hydrogen-Deuterium exchange kinetics in β-lactoglobulin (-)-epicatechin complexes studied by FTIR spectroscopy.

    Science.gov (United States)

    Caporaletti, Francesca; Carbonaro, Marina; Maselli, Paola; Nucara, Alessandro

    2017-11-01

    Hydrogen-Deuterium exchange kinetics of β-lactoglobulin and β-lactoglobulin (-)-epicatechin solutions has been investigated through the analysis of the amide I absorption band at 1650cm -1 in the FTIR spectrum. H-D substitution in NH amides and residues of the protein results in a slight red-shift and in intensity changes of the amide I components: either these effects have been inspected in the framework of the Principal Components Analysis methods. The present analysis allowed to unveil three H-D kinetics at the timescale of the oligomeric fluctuations of the protein. A fast mechanism (lifetime from 5 to 10min) can be ascribed to the dynamics of protein oligomers and aggregates at the scale of the quaternary structure variations, and it is not observed in the complexes β-lactoglobulin (-)-epicatechin. The other slowest kinetics, whose lifetimes are in the range 1-10h, are here associated to dynamics of high-molecular weight complexes that hamper the proton exchange. The role of (-)-epicatechin as an enhancer of the formation of stable high-molecular weight aggregates from β-lactoglobulin is also discussed by comparison of the lifetimes at different protein concentrations. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Rapid deuterium exchange-in time for probing conformational change

    International Nuclear Information System (INIS)

    Dharmasiri, K.; Smith, D.L.

    1995-01-01

    Isotopic exchange of protein backbone amide hydrogens has been used extensively as a sensitive probe of protein structure. One of the salient features of hydrogen exchange is the vast range of exchange rates in one protein. Isotopic exchange methods have been used to study the structural features including protein folding and unfolding (1), functionally different forms of proteins (2), protein-protein complexation (3), and protein stability parameter. Many backbone amide protons that are surface accessible and are not involved in hydrogen bonding undergo rapid deuterium exchange. In order to study, fast exchanging amide protons, fast exchange-in times are necessary

  10. Water-hydrogen isotope exchange process analysis

    International Nuclear Information System (INIS)

    Fedorchenko, O.; Alekseev, I.; Uborsky, V.

    2008-01-01

    The use of a numerical method is needed to find a solution to the equation system describing a general case of heterogeneous isotope exchange between gaseous hydrogen and liquid water in a column. A computer model of the column merely outputting the isotope compositions in the flows leaving the column, like the experimental column itself, is a 'black box' to a certain extent: the solution is not transparent and occasionally not fully comprehended. The approximate analytical solution was derived from the ZXY-diagram (McCabe-Thiele diagram), which illustrates the solution of the renewed computer model called 'EVIO-4.2' Several 'unusual' results and dependences have been analyzed and explained. (authors)

  11. Using hydrogen-deuterium exchange to monitor protein structure in the presence of gold nanoparticles.

    Science.gov (United States)

    Wang, Ailin; Vo, Tam; Le, Vu; Fitzkee, Nicholas C

    2014-12-11

    The potential applications of protein-functionalized gold nanoparticles (AuNPs) have motivated many studies characterizing protein-AuNP interactions. However, the lack of detailed structural information has hindered our ability to understand the mechanism of protein adsorption on AuNPs. In order to determine the structural perturbations that occur during adsorption, hydrogen/deuterium exchange (HDX) of amide protons was measured for two proteins by NMR. Specifically, we measured both slow (5-300 min) and fast (10-500 ms) H/D exchange rates for GB3 and ubiquitin, two well-characterized proteins. Overall, amide exchange rates are very similar in the presence and absence of AuNPs, supporting a model where the adsorbed protein remains largely folded on the AuNP surface. Small differences in exchange rates are observed for several loop residues, suggesting that the secondary structure remains relatively rigid while loops and surface residues can experience perturbations upon binding. Strikingly, several of these residues are close to lysines, which supports a model where positive surface residues may interact favorably with AuNP-bound citrate. Because these proteins appear to remain folded on AuNP surfaces, these studies suggest that it may be possible to engineer functional AuNP-based nanoconjugates without the use of chemical linkers.

  12. Exchange of bonded hydrogen in amorphous silicon by deuterium

    International Nuclear Information System (INIS)

    Abeles, B.; Yang, L.; Leta, D.P.; Majkrazak, C.

    1987-01-01

    The authors show that bonded hydrogen in a-Si:H is readily exchanged by atomic deuterium when exposed to a deuterium plasma discharge. The effective diffusion coefficient for the D, H exchange, 10 -14 cm 2 /sec at 160 0 C, is comparable to that of interstitial hydrogen in c-Si

  13. Investigation of hydrogen isotopic exchange catalysed by palladium phosphine complexes

    International Nuclear Information System (INIS)

    Zudin, V.N.; Rogov, V.A.; Likholobov, V.A.

    1985-01-01

    Basic regularities of the isotopic exchange reaction between molecular and protonated forms of hydrogen in the palladium phosphine complex system in CF 3 COOH aqueous solutions are studied by the radiochemical and mass-spectrometric methods using deuterium and tritium isotopes. The influences of C 2 H 4 and CO presence on the reaction proceeding are also studied by the same methods. It is established that bis(triphenyl phosphine)-bis(trifluoroacetate) palladium acts as a catalyst for hydrogen isotopic exchange. Hydrogen exchange runs through Pd hydride complex synthesis and decomposition, the hydrogen atom being capable of exchanging with solvent protons. Ethylene introduction into the system reduces the rate of the hydrogen exchange reaction, and the presence of the ethylene mixture and the carbon oxide inhibited it completely

  14. Hydrogen exchange dynamics of the P22 virion determined by time-resolved Raman spectroscopy. Effects of chromosome packaging on the kinetics of nucleotide exchanges.

    Science.gov (United States)

    Reilly, K E; Thomas, G J

    1994-08-05

    We describe the application of laser Raman spectroscopy to probe hydrogen isotope exchange dynamics of nucleic acid and protein constituents in a double-stranded DNA virus, the icosahedral bacteriophage P22. The Raman dynamic method employs a dialysis flow cell to control D2O efflux into an H2O solution of the virus sample while the rates of deuterium exchange of protons in the viral nucleic acid and protein molecules are measured spectrophotometrically in real time. The method provides structural and kinetic information about three different and distinct classes of exchangeable protons of the native virion: (1) labile imino (NH) and amino (NH2) protons of the bases which participate in Watson-Crick hydrogen bonding in the packaged genome; (2) pseudolabile purinic (8CH) protons that line the major groove of packaged P22 DNA; and (3) main-chain amide (NH) protons of viral subunits comprising the shell that encapsidates the DNA. The results obtained on P22 demonstrate that interchange of aqueous solvent with the virion interior is rapid and complete. We find that while labile protons of packaged DNA exchange rapidly, most amide protons in capsid subunits are resistant to solvent-catalyzed exchange. Further, stereospecific retardation of exchange is observed for major-groove protons of the packaged P22 genome. The quantitative measurements can be summarized and interpreted as follows. (1) Imino and amino protons of all bases in packaged P22 DNA exchange more rapidly (approximately 2-fold faster) than the corresponding protons in unpackaged P22 DNA. Remarkably, packaging actually accelerates labile imino and amino hydrogen exchanges of the viral DNA, an effect which can be attributed to selective stabilization in the packaged chromosome of a base-pair open state (breathing model). (2) Conversely, purine 8CH exchange rates in packaged P22 DNA are significantly retarded in comparison to those of unpackaged P22 DNA. The observed 8CH exchange retardation effects are

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

    Science.gov (United States)

    Yan, Xuguang; Zhang, Heidi; Watson, Jeffrey; Schimerlik, Michael I.; Deinzer, Max L.

    2002-01-01

    Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSFβ), 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-CSFβ) has fast and moderately fast exchangeable sections of amide hydrogens in the αA helix, and mostly slow exchanging sections of amide hydrogens in the αB, αC, and αD helices. Most of the amide hydrogens in the loop between the β1 and β4 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-CSFα than with the average B-factor. The rates of H/D exchange in rhM-CSFβ appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the αD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150–221 present in rhM-CSFβ, but not rhM-CSFα, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the αD helix is due to interaction of the carboxy-terminal tail with this helical segment. PMID:12192067

  16. Ligand assisted carbon dioxide activation and hydrogenation using molybdenum and tungsten amides.

    Science.gov (United States)

    Chakraborty, Subrata; Blacque, Olivier; Berke, Heinz

    2015-04-14

    The hepta-coordinated isomeric M(NO)Cl3(PN(H)P) complexes {M = Mo, ; W, , PN(H)P = (iPr2PCH2CH2)2NH, (HN atom of PN(H)P syn and anti to the NO ligand)} and the paramagnetic species M(NO)Cl2(PN(H)P) (M = Mo, ; W, ) could be prepared via a new synthetic pathway. The pseudo trigonal bipyramidal amides M(NO)(CO)(PNP) {M = Mo, ; W, ; [PNP](-) = [(iPr2PCH2CH2)2N](-)} were reacted with CO2 at room temperature with CO2 approaching the M[double bond, length as m-dash]N double bond in the equatorial (CO,NO,N) plane trans to the NO ligand and forming the pseudo-octahedral cyclic carbamates M(NO)(CO)(PNP)(OCO) (M = Mo, ; W = ). DFT calculations revealed that the approach to form the isomer is kinetically determined. The amine hydrides M(NO)H(CO)(PN(H)P) {M = Mo, ; W, }, obtained by H2 addition to , insert CO2 (2 bar) at room temperature into the M-H bond generating isomeric mixtures of the η(1)-formato complexes M(NO)(CO)(PN(H)P)(η(1)-OCHO), (M = Mo, ; M = W, ). Closing the stoichiometric cycles for sodium formate formation the isomeric mixtures were reacted with 1 equiv. of Na[N(SiMe3)2] regenerating . Attempts to turn the stoichiometric formate production into catalytic CO2 hydrogenation using in the presence of various types of sterically congested bases furnished yields of formate salts of up to 4%.

  17. Hydrogen-deuterium exchange in imidazole as a tool for studying histidine phosphorylation.

    Science.gov (United States)

    Cebo, Małgorzata; Kielmas, Martyna; Adamczyk, Justyna; Cebrat, Marek; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2014-12-01

    Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis. In this work, we investigated the effect of phosphorylation of the histidine side chain in peptides on deuterium-hydrogen exchange (DHX) in the imidazole. The results demonstrate that phosphorylation dramatically slows the rate of the DHX reaction. This phenomenon can be applied to detect phosphorylation of peptides at the histidine residue (e.g., in enzymatic digests). We also found that the influence of the peptide sequence on the exchange kinetics is relatively small. A CID fragmentation experiment revealed that there was no detectable hydrogen scrambling in peptides deuterated at C2 of the imidazole ring. Therefore, MS/MS can be used to directly identify the locations of deuterium ions incorporated into peptides containing multiple histidine moieties.

  18. Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR

    Science.gov (United States)

    Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

    2017-10-01

    In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

  19. Real-time hydrogen/deuterium exchange kinetics via supercharged electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Sterling, Harry J; Williams, Evan R

    2010-11-01

    Amide hydrogen/deuterium exchange (HDX) rate constants of bovine ubiquitin in an ammonium acetate solution containing 1% of the electrospray ionization (ESI) "supercharging" reagent m-nitrobenzyl alcohol (m-NBA) were obtained using top-down, electron transfer dissociation (ETD) tandem mass spectrometry (MS). The supercharging reagent replaces the acid and temperature "quench" step in the conventional MS approach to HDX experiments by causing rapid protein denaturation to occur in the ESI droplet. The higher charge state ions that are produced with m-NBA are more unfolded, as measured by ion mobility, and result in higher fragmentation efficiency and higher sequence coverage with ETD. Single amino acid resolution was obtained for 44 of 72 exchangeable amide sites, and summed kinetic data were obtained for regions of the protein where adjacent fragment ions were not observed, resulting in an overall spatial resolution of 1.3 residues. Comparison of these results with previous values from NMR indicates that the supercharging reagent does not cause significant structural changes to the protein in the initial ESI solution and that scrambling or back-exchange is minimal. This new method for top-down HDX-MS enables real-time kinetic data measurements under physiological conditions, similar to those obtained using NMR, with comparable spatial resolution and significantly better sensitivity.

  20. Deglycosylation induces extensive dynamics changes in α-amylase revealed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Ji, Chengjie; Wei, Gary

    2013-12-15

    N-Linked glycosylation plays important roles in modulating protein structure and function. The direct impact of the modification on protein conformation is not yet well understood. Here we probed the dynamic changes following Endo H trimming of high mannose glycans in α-amylase by means of amide hydrogen/deuterium exchange mass spectrometry. The results revealed that deglycosylation elicited extensive alterations in backbone dynamics, affecting regions both adjacent to and distal from the glycosylation site. The overall exchange rate is reduced in the glycosylated state, which indicates rigidity enhancement due to the attached carbohydrates. Copyright © 2013 John Wiley & Sons, Ltd.

  1. Trends in the exchange current for hydrogen evolution

    DEFF Research Database (Denmark)

    Nørskov, Jens Kehlet; Bligaard, Thomas; Logadottir, Ashildur

    2005-01-01

    A density functional theory database of hydrogen chemisorption energies on close packed surfaces of a number of transition and noble metals is presented. The bond energies are used to understand the trends in the exchange current for hydrogen evolution. A volcano curve is obtained when measured...... exchange currents are plotted as a function of the calculated hydrogen adsorption energies and a simple kinetic model is developed to understand the origin of the volcano. The volcano curve is also consistent with Pt being the most efficient electrocatalyst for hydrogen evolution. (c) 2005...... The Electrochemical Society. [DOI: 10.1149/1.1856988] All rights reserved....

  2. Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry

    DEFF Research Database (Denmark)

    Amon, Sabine; Trelle, Morten B; Jensen, Ole N

    2012-01-01

    Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions....... After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired......, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully...

  3. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Chalmers, Michael J; Busby, Scott A; Pascal, Bruce D; He, Yuanjun; Hendrickson, Christopher L; Marshall, Alan G; Griffin, Patrick R

    2006-02-15

    Amide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited its impact on drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D exchange system that provides highly reproducible H/D exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPARgamma induced upon binding a full agonist and a partial agonist. PPARgamma is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPARgamma have insulin sensitization properties while lacking several adverse effects associated with full agonist drugs. To further examine the mechanism of partial agonist activation of PPARgamma, we extended our studies to the analysis of ligand interactions with the heterodimeric complex of PPARgamma/RXRalpha LBDs. To facilitate analysis of H/D exchange of large protein complexes, we performed the experiment with a 14.5-T Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the ppb range.

  4. Isotopic hydrogen exchange in purines - mechanisms and applications

    International Nuclear Information System (INIS)

    Jones, J.R.; Taylor, S.E.

    1981-01-01

    The subject is reviewed under the headings: introduction (importance of purines in chemistry and biochemistry, and application of deuterium and tritium labelling); historical aspects (of purine labelling); experimental methods (use of detritiation methods to follow hydrogen isotope exchange in purines); rate - pH profiles (for isotopic hydrogen exchange); zwitterionic contributions; sites of protonation; metal-ion effects; magnitude of electrostatic factors; ionization constants; miscellaneous and concluding remarks. (U.K.)

  5. Isotope exchange between gaseous hydrogen and uranium hydride powder

    International Nuclear Information System (INIS)

    Shugard, Andrew D.; Buffleben, George M.; Johnson, Terry A.; Robinson, David B.

    2014-01-01

    Highlights: • Isotope exchange between hydrogen gas and uranium hydride powder can be rapid and reversible. • Gas–solid exchange rate is controlled by transport within ∼0.7 μm hydride particles. • Gas chromatographic separation of hydrogen isotopes using uranium hydride is feasible. - Abstract: Isotope exchange between gaseous hydrogen and solid uranium hydride has been studied by flowing hydrogen (deuterium) gas through packed powder beds of uranium deuteride (hydride). We used a residual gas analyzer system to perform real-time analysis of the effluent gas composition. We also developed an exchange and transport model and, by fitting it to the experimental data, extracted kinetic parameters for the isotope exchange reaction. Our results suggest that, from approximately 70 to 700 kPa and 25 to 400 °C, the gas-to-solid exchange rate is controlled by hydrogen and deuterium transport within the ∼0.7 μm diameter uranium hydride particles. We use our kinetic parameters to show that gas chromatographic separation of hydrogen and deuterium using uranium hydride could be feasible

  6. Heterogeneous Catalysis: Deuterium Exchange Reactions of Hydrogen and Methane

    Science.gov (United States)

    Mirich, Anne; Miller, Trisha Hoette; Klotz, Elsbeth; Mattson, Bruce

    2015-01-01

    Two gas phase deuterium/hydrogen exchange reactions are described utilizing a simple inexpensive glass catalyst tube containing 0.5% Pd on alumina through which gas mixtures can be passed and products collected for analysis. The first of these exchange reactions involves H[subscript 2] + D[subscript 2], which proceeds at temperatures as low as 77…

  7. Hydrogen and chlorine isotope exchange in n-methylimidazolium chloride

    International Nuclear Information System (INIS)

    Szydlowski, J.; Kimizuka, W.

    1993-01-01

    Isotope exchange of deuterium and 36 Cl between N-methylimidazolium chloride and gaseous hydrogen chloride has been studied over the temperature range of 249-322 K. A mechanism of exchange for both atoms is proposed and the equilibrium isotope effect of deuterium accompanying this reaction is discussed. (author) 10 refs.; 1 tab

  8. Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange

    DEFF Research Database (Denmark)

    Rand, Kasper Dyrberg; Jørgensen, Thomas; Olsen, Ole H

    2006-01-01

    Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics...

  9. Improved protein hydrogen/deuterium exchange mass spectrometry platform with fully automated data processing.

    Science.gov (United States)

    Zhang, Zhongqi; Zhang, Aming; Xiao, Gang

    2012-06-05

    Protein hydrogen/deuterium exchange (HDX) followed by protease digestion and mass spectrometric (MS) analysis is accepted as a standard method for studying protein conformation and conformational dynamics. In this article, an improved HDX MS platform with fully automated data processing is described. The platform significantly reduces systematic and random errors in the measurement by introducing two types of corrections in HDX data analysis. First, a mixture of short peptides with fast HDX rates is introduced as internal standards to adjust the variations in the extent of back exchange from run to run. Second, a designed unique peptide (PPPI) with slow intrinsic HDX rate is employed as another internal standard to reflect the possible differences in protein intrinsic HDX rates when protein conformations at different solution conditions are compared. HDX data processing is achieved with a comprehensive HDX model to simulate the deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer and enables fully unattended analysis of the entire protein HDX MS data set starting from ion detection and peptide identification to final processed HDX output, typically within 1 day. The final output of the automated data processing is a set (or the average) of the most possible protection factors for each backbone amide hydrogen. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

  10. Operating experiences of Heavy Water Plant at Talcher using bithermal ammonia-hydrogen exchange process (Paper No. 1.9)

    International Nuclear Information System (INIS)

    Haldar, T.K.; Manoj Kumar; Ramamurty, C.B.

    1992-01-01

    A heavy water plant employing bithermal ammonia-hydrogen exchange process was set up at Talcher (India). The energy consumption in the process is lower as compared to monothermal process. The plant performance was affected by various problems and has resulted in low heavy water production. The problems are: (i)formation of solid deposits, (ii)inability of exchange towers to process design gas flow rates, (iii)inadequate exchange efficiency of the sieve trays provided as exchange trays, and (iv)differential amide concentration in different plant sections. These problems and their effect on the overall plant performance are discussed in detail and modifications to improve the performance are suggested. (author). 4 figs

  11. Understanding hydrogen sorption in a metal-organic framework with open-metal sites and amide functional groups

    KAUST Repository

    Pham, Tony T.

    2013-05-09

    Grand canonical Monte Carlo (GCMC) studies of the mechanism of hydrogen sorption in an rht-MOF known as Cu-TPBTM are presented. The MOF is a decorated/substituted isostructural analogue to the unembellished rht-MOF, PCN-61, that was studied previously [ Forrest, K. A.J. Phys. Chem. C 2012, 116, 15538-15549. ]. The simulations were performed using three different hydrogen potentials of increasing complexity. Simulated hydrogen sorption isotherms and calculated isosteric heat of adsorption, Qst, values were in excellent agreement with the reported experimental data for only a polarizable model in one of four experimentally observed crystal structure configurations. The study demonstrates the ability of modeling to distinguish the differential sorption of distinct strucures; one configuration is found to be dominant due to favorable interactions with substrates. In addition, it was discovered that the presence of polar amide groups had a significant effect on the electrostatics of the Cu2+ ions and directs the low-pressure physisorption of hydrogen in the MOF. This is in contrast to what was observed in PCN-61, where an exterior copper ion had a higher relative charge and was the favored loading site. This tunability of the electrostatics of the copper ions via chemical substitution on the MOF framework can be explained by the presence of the negatively charged oxygen atom of the amide group that causes the interior Cu2+ ion to exhibit a higher positive charge through an inductive effect. Further, control simulations, taking advantage of the flexibility afforded by theoretical modeling, include artificially modified charges for both Cu2+ ions chosen equal to or with a higher charge on the exterior Cu2+ ion. This choice resulted in distinctly different hydrogen sorption characteristics in Cu-TPBTM with no direct sorption on the open-metal sites. Thus, this study demonstrates both the tunable nature of MOF platforms and the possibility for rational design of sorption

  12. Hydrogen--deuterium exchanges in nucleosides and nucleotides. A mechanism for exchange of the exocyclic amino hydrogens of adenosine

    International Nuclear Information System (INIS)

    Cross, D.G.; Brown, A.; Fisher, H.F.

    1975-01-01

    The pH dependence of the apparent first-order rate constant for the exchange of the exocyclic amino hydrogens of adenosine with deuterium from the solvent was measured by stopped-flow ultraviolet spectroscopy. This dependence shows acid catalysis, base catalysis, and spontaneous exchange at neutral pH values. A study of the effect of several buffers on the rates of exchange reveals both general acid and general base catalytic behavior for the exchange process. We propose a general mechanism for the exchange which requires N-1 protonated adenosine as an intermediate for the acid-catalyzed exchange and amidine anion for the base-catalyzed exchange. In both cases the rate-limiting step is the base-catalyzed abstraction of a proton from the exocyclic amino moiety. Evaluation of the rate constants predicts the equilibrium for the exocyclic amino/imino tautomers to be 6.3 x 10 3 :1. (U.S.)

  13. Measuring the hydrogen/deuterium exchange of proteins at high spatial resolution by mass spectrometry: overcoming gas-phase hydrogen/deuterium scrambling.

    Science.gov (United States)

    Rand, Kasper D; Zehl, Martin; Jørgensen, Thomas J D

    2014-10-21

    Proteins are dynamic molecules that exhibit conformational flexibility to function properly. Well-known examples of this are allosteric regulation of protein activity and ligand-induced conformational changes in protein receptors. Detailed knowledge of the conformational properties of proteins is therefore pertinent to both basic and applied research, including drug development, since the majority of drugs target protein receptors and a growing number of drugs introduced to the market are therapeutic peptides or proteins. X-ray crystallography provides a static picture at atomic resolution of the lowest-energy structure of the native ensemble. There is a growing need for sensitive analytical tools to explore all of the significant molecular structures in the conformational landscape of proteins. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has recently emerged as a powerful method for characterizing protein conformational dynamics. The basis of this method is the fact that backbone amides in stable hydrogen-bonded structures (e.g., α-helices and β-sheets) are protected against exchange with the aqueous solvent. All protein structures are dynamic, however, and eventually all of the protecting hydrogen bonds will transiently break as the protein--according to thermodynamic principles--cycles through partially unfolded states that correspond to excited free energy levels. As a result, all of the backbone amides will eventually become temporarily solvent-exposed and exchange-competent over time. Consequently, a folded protein in D2O will gradually incorporate deuterium into its backbone amides, and the kinetics of the process can be readily monitored by mass spectrometry. The deuterium uptake kinetics for the intact protein (global exchange kinetics) represents the sum of the exchange kinetics for the individual backbone amides. Local exchange kinetics is typically achieved by using pepsin digestion under quench conditions (i.e., under cold

  14. Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry*

    Science.gov (United States)

    Stokasimov, Ema; Rubenstein, Peter A.

    2009-01-01

    Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts. PMID:19605362

  15. Preparation of deuterium-labeled rutin by hydrogen exchange reaction

    International Nuclear Information System (INIS)

    Hiraoka, K.; Miyamoto, T.

    1981-01-01

    Preparation of deuterium-labeled rutin by hydrogen exchange reaction under alkaline condition is described. Hydrogens at positions 2',5' and 6' of rutin were replaced with deuteriums only on heating, while hydrogens at positions 6 and 8 were readily replaced at room temperature. On the basis of these findings rutin-2',5',6',6,8-d 5 was first prepared and then treated with alkaline water at room temperature to obtain rutin labeled with deuteriums at positions 2',5' and 6'. (author)

  16. Deuterium separation factors in ammonia-hydrogen exchange

    International Nuclear Information System (INIS)

    Dave, S.M.; Sadhukhan, H.K.; Raman, S.; Srisaila, S.; Kalsi, A.S.

    1978-01-01

    Deuterium separation factors for the exchange between ammonia and hydrogen involving all the isotopic forms of these molecules at equilibrium have been calculated as function of temperature and deuterium concentration, using recent spectroscopic data. Wide range of temperature and deuterium concentration has been covered. These calculated values are in excellent agreement with the experimental measurements reported by different workers. (author)

  17. Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Li, Sheng; Lee, Su Youn; Chung, Ka Young

    2015-01-01

    Conformational change and protein-protein interactions are two major mechanisms of membrane protein signal transduction, including G protein-coupled receptors (GPCRs). Upon agonist binding, GPCRs change conformation, resulting in interaction with downstream signaling molecules such as G proteins. To understand the precise signaling mechanism, studies have investigated the structural mechanism of GPCR signaling using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in GPCR studies. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and excluded or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein-protein interfaces and conformational changes after protein activation or protein-protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. Interactions between GPCRs and downstream signaling molecules are not easily analyzed by X-ray crystallography or NMR because of the large size of the GPCR-signaling molecule complexes, hydrophobicity, and flexibility of GPCRs. HDX-MS could be useful for analyzing the conformational mechanism of GPCR signaling. In this chapter, we discuss details of HDX-MS for analyzing GPCRs using the β2AR-G protein complex as a model system. © 2015 Elsevier Inc. All rights reserved.

  18. Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics.

    Science.gov (United States)

    Mazon, Hortense; Marcillat, Olivier; Forest, Eric; Vial, Christian

    2004-02-01

    Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism.

  19. Isotopic exchange of hydrogen in aromatic amino acids

    International Nuclear Information System (INIS)

    Pshenichnikova, A.B.; Karnaukhova, E.N.; Mitsner, B.I.

    1993-01-01

    The kinetics of the isotopic replacement of hydrogen in the aromatic amino acids L-tryptophan, L-tyrosine, and L-phenylalanine in solutions of deuterochloric and deuterosulfuric acids in deuterium oxide were investigated by PMR spectroscopy. The reactions were shown to be of first orders with respect both to the concentration of the substrate and to the activity of the deuterium ion. The isotopic effects of hydrogen and the values of the activation energy of H-D exchange in different positions of the aromatic ring in tryptophan and tyrosine were determined. The effect of properties of the medium on the rate of the isotopic exchange of hydrogen is discussed. 17 refs., 2 figs., 2 tabs

  20. Quantum nonadiabatic dynamics of hydrogen exchange reactions

    Science.gov (United States)

    Rajagopala Rao, T.; Jayachander Rao, B.; Mahapatra, S.

    2009-11-01

    In continuation of our earlier effort to understand the nonadiabatic coupling effects in the prototypical H + H 2 exchange reaction [Jayachander Rao et al. Chem. Phys. 333 (2007) 135], we present here further quantum dynamical investigations on its isotopic variants. The present work also corrects a technical scaling error occurred in our previous studies on the H + HD reaction. Initial state-selected total reaction cross sections and Boltzmann averaged thermal rate constants are calculated with the aid of a time-dependent wave packet approach employing the double many body expansion potential energy surfaces of the system. The theoretical results are compared with the experimental and other theoretical data whenever available. The results re-establish our earlier conclusion, on a more general perspective, that the electronic nonadiabatic effects are negligible on the important quantum dynamical observables of these reactive systems reported here.

  1. Quantum nonadiabatic dynamics of hydrogen exchange reactions

    International Nuclear Information System (INIS)

    Rajagopala Rao, T.; Jayachander Rao, B.; Mahapatra, S.

    2009-01-01

    In continuation of our earlier effort to understand the nonadiabatic coupling effects in the prototypical H + H 2 exchange reaction [Jayachander Rao et al. Chem. Phys. 333 (2007) 135], we present here further quantum dynamical investigations on its isotopic variants. The present work also corrects a technical scaling error occurred in our previous studies on the H + HD reaction. Initial state-selected total reaction cross sections and Boltzmann averaged thermal rate constants are calculated with the aid of a time-dependent wave packet approach employing the double many body expansion potential energy surfaces of the system. The theoretical results are compared with the experimental and other theoretical data whenever available. The results re-establish our earlier conclusion, on a more general perspective, that the electronic nonadiabatic effects are negligible on the important quantum dynamical observables of these reactive systems reported here.

  2. On mechanism of water-hydrogen isotope exchange reaction

    International Nuclear Information System (INIS)

    Iida, Itsuo

    1982-01-01

    The catalyst for water-hydrogen isotope exchange reaction for heavy water concentration, such as Pt-charcoal and Ni-chromia, has not been sufficient in the activity at low temperature. However, the catalyst of platinum carried by hydrophobic polymers was highly active in the water-hydrogen isotope exchange reaction at room temperature, and the fact that the catalyst is not wet has an important significance for the activity in low temperature region. However, the physical and chemical meanings of ''not wet'' in this system are not necessarily clear. In this study, hydrophilic catalyst with alumina carrier and hydrophobic catalysts with PTFE and other carriers were tested in hydrogen-steam isotope exchange reaction and the adsorption of water, and the physical and chemical meanings of ''not wet'' in isotope exchange reaction were examined. The experiment and its results are reported. Since water is adsorbed intensely on alumina, multi-molecular layer adsorption occurs, and platinum particles are buried under water, and lose the activity. In case of the platinum catalyst carried by hydrophobic carriers, only the water of single molecule layer is adsorbed on platinum, therefore, the activity is not lost. (Kako, I.)

  3. Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Yang, Sheng-Wei; Ting, Hsiu-Chi; Lo, Yi-Ting; Wu, Ting-Yuan; Huang, Hung-Wei; Yang, Chia-Jung; Chan, Jui-Fen Riva; Chuang, Min-Chieh; Hsu, Yuan-Hao Howard

    2016-01-01

    Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. O--H charge exchange in cold, dense, hydrogen plasmas

    International Nuclear Information System (INIS)

    Cohen, S.A.; Dylla, H.F.

    1977-05-01

    It is pointed out that the accidentally resonant charge exchange reaction, O + + H 0 reverse arrows O 0 + H + , is an important mechanism for causing the loss of singly charged oxygen ions from oxygen contaminated hydrogen plasmas. Results of a Monte Carlo simulation are presented which show that the fraction of oxygen lost because of charge exchange exceeds 1 / 3 when the parameters n/sub e/ approx. 10 13 cm -3 , n/sub H//sup o/ approx. 10 11 cm -3 and T/sub e/ approx. 3 eV are attained

  5. Hydrogen-deuterium exchange study of an allosteric energy cycle.

    Science.gov (United States)

    Beckett, Dorothy

    2012-01-01

    Elucidation of mechanisms of energy transduction through macromolecules in allosteric systems requires application of a broad range of techniques and approaches. High-resolution structures of the end states in an allosteric system provide invaluable clues about allosteric mechanism. Thermodynamic and kinetic studies reveal the rules that govern the transitions between states in the system. Acquisition of detailed molecular level information about allosteric mechanism requires interrogation of the structural and dynamic properties of both intermediates and end states in the allosteric cycle. Many experimental and computational tools have been developed to probe allostery. Among these are hydrogen-deuterium exchange detected by either NMR spectroscopy or mass spectrometry. This article provides a detailed description of application of hydrogen exchange detected by mass spectrometry (HDX-MS) to investigate an allosteric system.

  6. An overview of hydrogen deuterium exchange mass spectrometry (HDX-MS) in drug discovery.

    Science.gov (United States)

    Masson, Glenn R; Jenkins, Meredith L; Burke, John E

    2017-10-01

    Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein dynamics, protein folding, protein-protein interactions, and protein small molecule interactions. The development of novel methodologies and technical advancements in mass spectrometers has greatly expanded the accessibility and acceptance of this technique within both academia and industry. Areas covered: This review examines the theoretical basis of how amide exchange occurs, how different mass spectrometer approaches can be used for HDX-MS experiments, as well as the use of HDX-MS in drug development, specifically focusing on how HDX-MS is used to characterize bio-therapeutics, and its use in examining protein-protein and protein small molecule interactions. Expert opinion: HDX-MS has been widely accepted within the pharmaceutical industry for the characterization of bio-therapeutics as well as in the mapping of antibody drug epitopes. However, there is room for this technique to be more widely used in the drug discovery process. This is particularly true in the use of HDX-MS as a complement to other high-resolution structural approaches, as well as in the development of small molecule therapeutics that can target both active-site and allosteric binding sites.

  7. Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium-exchange to characterize allosteric regulation†

    Science.gov (United States)

    Prasannan, Charulata B.; Artigues, Antonio; Fenton, Aron W.

    2011-01-01

    There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g. active site and allosteric site). Both NMR, by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H2O and D2O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D2O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D2O, but the effector continues to elicit an allosteric response. PMID:21701851

  8. Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation.

    Science.gov (United States)

    Prasannan, Charulata B; Artigues, Antonio; Fenton, Aron W

    2011-08-01

    There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H(2)O and D(2)O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D(2)O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D(2)O, but the effector continues to elicit an allosteric response.

  9. Hydrogen/deuterium exchange in interstellar ice analogs

    Science.gov (United States)

    Ratajczak, A.; Quirico, E.; Faure, A.; Schmitt, B.; Ceccarelli, C.

    2009-03-01

    Context: For several reasons, methanol is believed to be formed on grain surfaces and, in warm environments, released in the gas phase. In the past, multiply deuterated isotopologues of methanol have been detected in gas phase around several low-mass protostars. In all these sources, there is significantly more CH2DOH than CH3OD. Various hypotheses have been suggested to explain this anomaly, but none is fully convincing. Aims: In this work, we test a new hypothesis experimentally: the spontaneous exchange between hydrogen and deuterium atoms in water ice as responsible for the deficiency of CH3OD with respect to CH2DOH. Methods: We follow the temperature dependence of the composition of interstellar ice analogs initially composed of CD3OD and H2O. To this aim, thin films of intimate H2O:CD3OD ice mixtures, condensed at low temperature (hydrogen/deuterium (H/D) exchange is observed, at 120 K and above, through the growth of the ν_OD stretching mode of HDO at ~2425 cm-1. It is also shown that H/D exchange occurs i) on the hydroxyl functional group of methanol, i.e through hydrogen bonds, and ii) before the completion of crystallization. Conclusions: The present results suggest that the much lower abundance of CH3OD compared to CH2DOH in low-mass protostars could reflect H/D exchanges in water ice either prior to or definitely during the grain mantle sublimation. This solid-state depletion mechanism, so far neglected in the astronomical literature, might affect other deuterated molecules with hydrogen bonds.

  10. Hydrogen isotope exchange reaction rates in tritium, hydrogen and deuterium mixed gases

    International Nuclear Information System (INIS)

    Uda, Tatsuhiko

    1992-01-01

    Hydrogen isotope exchange reaction rates in H 2 +T 2 , D 2 +T 2 and H 2 +D 2 +T 2 mixed gases, as induced by tritium decay and beta radiation, were experimentally measured by laser Raman spectrometry. Initially a glass cell was filled with T 2 gas to a pressure of 30-40 kPa, and an equivalent partial pressure of H 2 and/or D 2 was added. The first-order hydrogen isotope exchange reaction rates were 5.54x10 -2 h -1 for H 2 +T 2 mixed gas and 4.76x10 -2 h -1 for D 2 +T 2 . The actual HT producing rate was nearly equivalent to the rate of DT, but the reverse reaction rate of HT was faster than that of DT. The exchange reaction rates between H, D and T showed the isotope effect, HD>HT>DT. The hydrogen isotope exchange reaction rates observed were about twenty times larger than ion formation rates by beta radiation. This result suggests that a free radical chain reaction in hydrogen isotopes is occurring. (orig.)

  11. Subzero Celsius separations in three-zone temperature controlled hydrogen deuterium exchange mass spectrometry.

    Science.gov (United States)

    Wales, Thomas E; Fadgen, Keith E; Eggertson, Michael J; Engen, John R

    2017-11-10

    Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent. To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry. Separation at 0°C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases. Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time. Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures. A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing. To prevent freezing, 35% methanol is introduced post online digestion. No new pumps are required and online digestion is performed as in the past. Subzero separations, using conventional HPLC column geometry of 3μ m particles in a 1×50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20°C. There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems. Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Gas-phase hydrogen-deuterium exchange reactions in carbanions: exchange of vinyl and aryl protons by D2O

    International Nuclear Information System (INIS)

    Squires, R.R.; DePuy, C.H.; Bierbaum, V.M.

    1981-01-01

    This paper reports that under the proper conditions D 2 O can serve as an exchange reagent for hydrogens which are much less acidic than water; as a result, in certain carbanions aryl and vinyl hydrogens can be observed to exchange with D 2 O

  13. The unusual hydrogen-deuterium exchange of α-carbon protons in N-substituted glycine-containing peptides.

    Science.gov (United States)

    Bąchor, Remigiusz; Setner, Bartosz; Kluczyk, Alicja; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2014-01-01

    Hydrogens connected to α-carbon (α-C) of amino acid residues are usually resistant to hydrogen-deuterium exchange (HDX) unless reaction conditions promote racemization. Although N-methylglycine (sarcosine) residue has been found in biologically active peptide such as cyclosporine, to the best of our knowledge, the HDX of α-C protons of this residue was not explored yet. Here, we presented a new and efficient methodology of α-C deuteration in sarcosine residues under basic aqueous conditions. The deuterons, introduced at α-C atom, do not undergo back-exchange in acidic aqueous solution. The electrospray ionization-MS and MS/MS experiments on proposed model peptides confirmed the HDX at α-C and revealed the unexpected hydrogen scrambling in sarcosine-containing peptides. Although the observed HDX of α-C protons is only successful in N-acylglycine when the amide possesses a certain degree of alkylation, it offers a new approach to the analysis of sarcosine-containing peptides such as cyclosporine. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Spin exchange optical pumping of hydrogen and deuterium nuclei

    International Nuclear Information System (INIS)

    Anderson, L.W.; Walker, T.

    1995-01-01

    The prospects for the direct production of a nuclear polarized atomic hydrogen or deuterium target by the use of spin exchange optical pumping is analyzed in both a low and a high magnetic field. In a low magnetic field it is found that the optical pumping cell wall coatings must have relaxation times that correspond to thousands of wall collisions if one is to produce a highly polarized target. In a high magnetic field it is found that wall coatings must have relaxation times that correspond to only a few hundred wall collisions in order to produce a highly polarized atomic hydrogen target but must have relaxation times that correspond to thousands of wall collisions to produce a highly polarized atomic deuterium target. ((orig.))

  15. Optical activity studies of hydrogen-deuterium exchange

    International Nuclear Information System (INIS)

    Hansen, L.J.

    1990-01-01

    The potassium complexes of racemic and optically active forms of 1,2-propanediaminetriacetatoacetic acid nickel-ate (II) were prepared stoichiometrically by two different experimental procedures. The complexes were characterized by UV-VIS absorption spectroscopy, infrared spectroscopy, and thermal analysis. Circular dichroism and optical rotatory dispersion values were obtained on the optically active complexes. TGA and IR spectroscopy techniques suggest that Δ-K [Ni(R (-)HPDTA)] H 2 O (1)and Λ-K [Ni(S (+)HPDTA)] H 2 O (2) have different configurations in solution than in the solid state. Solid complexes of (1) are theorized to have the nickel (II) ion bound pentadentate to the PDTA ligand and unidentate to a water molecule. The free carboxyl arm of the PDTA ligand is protonated. Dissolution of the complexes results in rotational changes which occur with time. The rate of rotational change has been kinetically measured, which results in three pH dependent rate constants. An isotope effect for such reactions in H 2 O and D 2 O has been measured. The base-catalyzed hydrogen-deuterium exchange of the out-of-plane glycinate rings of (1) and (2) complexes has been determined for three of the four glycinate protons by ORD. The rate of hydrogen-deuterium exchange is extremely slow and consecutive proton exchanges are not independent of one another over sufficiently long periods, such that measurement of α ∞ are calculated by three differing mathematical models and applied to the calculation of the hydrogen-deuterium rate constants

  16. Hydrogen-tritium exchange of rhodopsin: effect of solvent on the incorporation of slowly exchanging tritium atoms

    International Nuclear Information System (INIS)

    Osborne, H.B.

    1976-01-01

    The hydrogen-tritium exchange technique has been used to demonstrate the presence of conformational changes in proteins. They are visualized as changes in the exchange kinetics of the proteins labile hydrogens. To enable the study of the conformational changes of rhodopsin - the visual pigment of the vertebrate retinal rod outer sigments - upon illumination, it is necessary to ensure that the associated labile hydrogens become tritiated during incubation time. The effect of several incubation media on the rhodopsin exchange-in-kinetics have been studied.. The solubilisation effect by detergent on the exchange-in-kinetics of rhodopsin was also investigated. It is shown that both membrane-bound and detergent-solubilised rhodopsin possess an important number of very slowly exchanging hydrogen atoms. The number of slowly exchanging tritium atoms incorporated in rhodopsin is greatly increased by the presence of phosphate ions in the incubation medium

  17. Chemical-exchange-sensitive MRI of amide, amine and NOE at 9.4 T versus 15.2 T.

    Science.gov (United States)

    Chung, Julius Juhyun; Choi, Wonmin; Jin, Tao; Lee, Jung Hee; Kim, Seong-Gi

    2017-09-01

    Chemical exchange (CE)-sensitive MRI benefits greatly from stronger magnetic fields; however, field effects on CE-sensitive imaging have not yet been studied well in vivo. We have compared CE-sensitive Z-spectra and maps obtained at the fields of 9.4 T and 15.2 T in phantoms and rats with off-resonance chemical-exchange-sensitive spin lock (CESL), which is similar to conventional chemical exchange saturation transfer. At higher fields, the background peak at water resonance has less spread and the exchange rate relative to chemical shift decreases, thus CESL intensity is dependent on B 0 . For the in vivo amide and nuclear Overhauser enhancement (NOE) composite resonances of rat brains, intensities were similar for both magnetic fields, but effective amide proton transfer and NOE values obtained with three-point quantification or a curve fitting method were larger at 15.2 T due to the reduced spread of attenuation at the direct water resonance. When using intermediate exchange-sensitive irradiation parameters, the amine proton signal was 65% higher at 15.2 T than at 9.4 T due to a reduced ratio of exchange rate to chemical shift. In summary, increasing magnetic field provides enhancements to CE-sensitive signals in the intermediate exchange regime and reduces contamination from background signals in the slow exchange regime. Consequently, ultrahigh magnetic field is advantageous for CE-sensitive MRI, especially for amine and hydroxyl protons. Copyright © 2017 John Wiley & Sons, Ltd.

  18. Hydrogen-Deuterium exchange monitored by ATR-FTIR spectroscopy

    Science.gov (United States)

    Poe, Brent; Del Vecchio, Alessandro; Cestelli Guidi, Mariangela

    2016-04-01

    Measuring the extent of isotopic exchange is a common means for the determination of self-diffusion coefficients in any type of medium (gas, liquid, amorphous solid, crystalline solid). For rapidly diffusing species such as hydrogen in condensed phases, real time methods involving vibrational spectroscopy can be used by taking advantage of the large relative mass difference between 1H and 2H, resulting in large differences in the band positions of their vibrational modes. We demonstrate rapid isotopic exchange between D2O liquid and H2O vapor using ATR (attenuated total reflectance) in a FTIR spectrometer. Over the course of a few minutes several spectra were acquired of a D2O droplet mounted on a diamond crystal. The progressive exchange reaction between the liquid phase and H2O from the atmosphere was monitored by measuring the decreasing absorbance of the D-O-D bending and O-D stretching bands and the increasing absorbance of the D-O-H and H-O-H bending and O-H stretching bands as functions of time. Our results offer some intriguing insights into the structural characteristics of water as inferred by this exchange process.

  19. Contribution to the study of catalytic hydrogen-deuterium exchange between hydrogen and hydrocarbons

    International Nuclear Information System (INIS)

    Ravoire, J.

    1958-01-01

    The hydrogen-deuterium exchange between molecular hydrogen and hydrocarbons over a platinum and charcoal catalyst was studied in a static system. The change in isotopic composition of molecular hydrogen was followed by a thermal conductivity method. Cyclo-pentane and cyclohexane were chosen because of their stability. A reversible inactivation of the catalyst was observed with both hydrocarbons. The reasons for this inactivation are unknown but it was shown that reactivation led to satisfactory reproducibility. A kinetic study was done with cyclohexane in the range 30 to 160 deg. C, and 40 to 360 mm for the pressure of hydrogen, and 10 to 70 mm for the pressure of cyclohexane. The order of the reaction with respect to cyclohexane pressure is always close to zero; the order with respect to that of hydrogen is 0.5 above 100 deg. C. It decreases with increasing temperature and becomes negative (-0.5 at 30 deg. C), characterizing an inhibition by hydrogen. At the same time, the apparent activation energy goes from 6 to 13 kcal/mole. (author) [fr

  20. Simple atmospheric hydrogen/deuterium exchange method for enumeration of labile hydrogens by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Nikolaev, Eugene

    2013-06-04

    A simple method for hydrogen/deuterium exchange in a standard electrospray (ESI) ionization source is presented. In this method, a D₂O droplet is placed between the ESI needle and the entrance of the mass spectrometer and thus saturation of the atmosphere with deuterated vapor in the ESI region is achieved. It was shown that full exchange of up to 23 labile acidic hydrogens with a minimal back exchange with the surrounding atmospheric water can be performed by this method.

  1. Effects of protein-ligand interactions on hydrogen/deuterium exchange kinetics: canonical and noncanonical scenarios.

    Science.gov (United States)

    Sowole, Modupeola A; Konermann, Lars

    2014-07-01

    Hydrogen/deuterium exchange (HDX) methods are widely used for monitoring protein-ligand interactions. This approach relies on the fact that ligand binding can modulate the extent of protein structural fluctuations that transiently disrupt hydrogen bonds and expose backbone amides to the solvent. It is commonly observed that ligand binding causes a reduction of HDX rates. This reduction can be restricted to elements adjacent to the binding site, but other regions can be affected as well. Qualitatively, ligand-induced HDX protection can be rationalized on the basis of two-state models that equate structural dynamics with global unfolding/refolding. Unfortunately, such models tend to be unrealistic because the dynamics of native proteins are dominated by subglobal transitions and local fluctuations. Ligand binding lowers the ground-state free energy. It is not obvious why this should necessarily be accompanied by a depletion of excited-state occupancies, which would be required for a reduction of HDX rates. Here, we propose a framework that implies that ligand binding can either slow or accelerate amide deuteration throughout the protein. These scenarios are referred to as "type 1" and "type 2", respectively. Evidence for type 1 binding is abundant in the literature, whereas the viability of type 2 interactions is less clear. Using HDX mass spectrometry (MS), we demonstrate that the oxygenation of hemoglobin (Hb) provides a dramatic example of a type 2 scenario. The observed behavior is consistent with cooperative T → R switching, where part of the intrinsic O2 binding energy is reinvested for destabilization of the ground state. This destabilization increases the Boltzmann occupancy of unfolded conformers, thereby enhancing HDX rates. Surprisingly, O2 binding to myoglobin (Mb) also induces elevated HDX rates. These Mb data reveal that type 2 behavior is not limited to cooperative multisubunit systems. Although enhanced protection from deuteration is widely

  2. Minimizing Back Exchange in the Hydrogen Exchange-Mass Spectrometry Experiment

    Science.gov (United States)

    Walters, Benjamin T.; Ricciuti, Alec; Mayne, Leland; Englander, S. Walter

    2012-12-01

    The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology to the study of large biologically important proteins. A persistent problem is the degradation of HX information due to back exchange of deuterium label during the fragmentation-separation process needed to prepare samples for MS measurement. This paper reports a systematic analysis of the factors that influence back exchange (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates, system volume). The many peptides exhibit a range of back exchange due to intrinsic amino acid HX rate differences. Accordingly, large back exchange leads to large variability in D-recovery from one residue to another as well as one peptide to another that cannot be corrected for by reference to any single peptide-level measurement. The usual effort to limit back exchange by limiting LC time provides little gain. Shortening the LC elution gradient by 3-fold only reduced back exchange by ~2 %, while sacrificing S/N and peptide count. An unexpected dependence of back exchange on ionic strength as well as pH suggests a strategy in which solution conditions are changed during sample preparation. Higher salt should be used in the first stage of sample preparation (proteolysis and trapping) and lower salt (<20 mM) and pH in the second stage before electrospray injection. Adjustment of these and other factors together with recent advances in peptide fragment detection yields hundreds of peptide fragments with D-label recovery of 90 % ± 5 %.

  3. Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment.

    Science.gov (United States)

    Walters, Benjamin T; Ricciuti, Alec; Mayne, Leland; Englander, S Walter

    2012-12-01

    The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology to the study of large biologically important proteins. A persistent problem is the degradation of HX information due to back exchange of deuterium label during the fragmentation-separation process needed to prepare samples for MS measurement. This paper reports a systematic analysis of the factors that influence back exchange (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates, system volume). The many peptides exhibit a range of back exchange due to intrinsic amino acid HX rate differences. Accordingly, large back exchange leads to large variability in D-recovery from one residue to another as well as one peptide to another that cannot be corrected for by reference to any single peptide-level measurement. The usual effort to limit back exchange by limiting LC time provides little gain. Shortening the LC elution gradient by 3-fold only reduced back exchange by ~2%, while sacrificing S/N and peptide count. An unexpected dependence of back exchange on ionic strength as well as pH suggests a strategy in which solution conditions are changed during sample preparation. Higher salt should be used in the first stage of sample preparation (proteolysis and trapping) and lower salt (<20 mM) and pH in the second stage before electrospray injection. Adjustment of these and other factors together with recent advances in peptide fragment detection yields hundreds of peptide fragments with D-label recovery of 90% ± 5%.

  4. Deuterium-hydrogen isotopic exchange in water molecules adsorbed on Teflon under atomic-molecular hydrogen beams

    International Nuclear Information System (INIS)

    Grankin, V.P.; Savinkov, N.A.; Styrov, V.V.; Tyurin, Yu.I.

    1994-01-01

    Processes of deuterium-hydrogen exchange in the course of interaction between hydrogen molecular beam and H+H 2 atomic-molecular beam with adsorbed water molecules from D 2 O, HDO, H 2 O on Teflon have been studied. Desorption of the above molecules into vacuum, as well as their desorption under conditions of molecular and atomic-molecular hydrogen beam effect on Teflon surface have been investigated experimentally. Relative probabilities of hydrogen isotopes desorption from Teflon surface have been defined, relative probabilities and cross sections of diverse reactions of isotopic exchange have been found. 2 refs.; 3 figs

  5. Considerations in the analysis of hydrogen exchange mass spectrometry data

    Science.gov (United States)

    Wales, Thomas E.; Eggertson, Michael J.; Engen, John R.

    2013-01-01

    i. Summary A major component of a hydrogen exchange mass spectrometry experiment is the analysis of protein and peptide mass spectra to yield information about deuterium incorporation. The processing of data that are produced includes the identification of each peptic peptide to create a master table/array of peptide sequence, retention time and retention time range, mass range and undeuterated mass. The amount of deuterium incorporated into each of the peptides in this array must then be determined. Various software platforms have been developed in order to perform this specific type of data analysis. We describe the fundamental parameters to be considered at each step along the way and how data processing, either by an individual or by software, must approach the analysis. PMID:23666730

  6. Hydrogen-Deuterium Exchange Profiles of Polyubiquitin Fibrils

    Directory of Open Access Journals (Sweden)

    Daichi Morimoto

    2018-02-01

    Full Text Available Ubiquitin and its polymeric forms are conjugated to intracellular proteins to regulate diverse intracellular processes. Intriguingly, polyubiquitin has also been identified as a component of pathological protein aggregates associated with Alzheimer’s disease and other neurodegenerative disorders. We recently found that polyubiquitin can form amyloid-like fibrils, and that these fibrillar aggregates can be degraded by macroautophagy. Although the structural properties appear to function in recognition of the fibrils, no structural information on polyubiquitin fibrils has been reported so far. Here, we identify the core of M1-linked diubiquitin fibrils from hydrogen-deuterium exchange experiments using solution nuclear magnetic resonance (NMR spectroscopy. Intriguingly, intrinsically flexible regions became highly solvent-protected in the fibril structure. These results indicate that polyubiquitin fibrils are formed by inter-molecular interactions between relatively flexible structural components, including the loops and edges of secondary structure elements.

  7. Toward standardizing deuterium content reporting in hydrogen exchange-MS.

    Science.gov (United States)

    Sheff, Joey G; Schriemer, David C

    2014-12-16

    We introduce a method to monitor dispensing ratios during labeling reactions in hydrogen exchange (HX)-MS. The method corrects for systematic and random dispensing errors and harmonizes data incorporating variable %D2O in the experiment design. A correction factor for deuterium levels is obtained by quantifying the relative signal intensities arising from nonexchanging heavy caffeine (spiked into labeling buffer) and light caffeine (spiked into sample solutions). Dispensing variability over a wide range of %D2O composition can be detected and corrected to a common value, and although random dispensing error is usually minor, we show it can be the limiting factor in high quality signal measurements. Applying a dispensing control is therefore an effective tool for monitoring measurement precision in HX-MS.

  8. Hydrogen isotope exchange experiments with Mt Mazama ash

    Science.gov (United States)

    Nolan, G. S.; Bindeman, I. N.; Palandri, J. L.

    2011-12-01

    minor (~20%) hydroxyl loss. 5) Native ash dried to exchange through time and modeling the reaction as (pseudo) first order in concentration of deuterium yields the following isotopic half life values: for 70° C 1.43 years, for 40° C 4.1 years, and for 25 °C 12.1 years. An Arrhenius treatment yields an activation energy of 32-38 kjoules. 6) A second order kinetic treatment can be used to follow the reaction. A calculation using the reaction constant k at 25 °C assuming native ash exposed to -60 % ∂2H water indicates it would take 2 years to rise from a starting value of -150 to approximately -100 % ∂2H. Results of these experiments put quantitative limits on reliability of ∂2H in ash in paleo-climate studies that are primarily controlled by the isotopic environments and temperatures. We hypothesize that hydrogen-deuterium exchange occurs in native ash exposed to isotopically labeled water. The effect is mediated through surface correlated water in addition to diffusion with both having a rate determining effect. There is nothing to suggest that deuterium-hydrogen exchange is selective for molecular or SiOH hydrogen in the ash.

  9. Investigation of hydrogen exchange for deuterium and tritium in α-aminoalkylphosphonous acids

    International Nuclear Information System (INIS)

    Pokrovskaya, M.Yu.; Shumyantseva, V.V.; Lesnik, E.A.

    1990-01-01

    Kinetics of hydrogen isotopic exchange for deuterium in α-aminoalkylphosphonous acids depending on pH using PMR-spectroscopy is investigated. Tritium labelled α-aminoalkylphosphonous acids have resulted from direct hydrogen-tritium exchange between T 2 O and hydrophosphoryl compounds

  10. Hydrogen isotope exchange experiments with Mt Mazama ash

    Science.gov (United States)

    Nolan, G. S.; Bindeman, I. N.; Palandri, J. L.

    2010-12-01

    In recent years the D/H ratio in hydrous minerals and volcanic glass has been used to evaluate δD value of coexisting meteoric water and their ability to preserve environmental conditions through time. We report a series of D-H exposure experiments of ash at 70, 40 and 25°C varying in time from 0 to >2000 h in order to evaluate the facility of hydrogen uptake from deuterated waters (650‰ to pure D2O). Measurements were performed on 1-3 mg of ash using TCEA-MAT253 GSMS. Time series experiments aided by infrared measurements demonstrate the following new results: 1) We observe 5 to 90‰ deuterium uptake depending on exposure time and temperature with uptake positively correlated with temperature. Increases by ~5‰ δD are seen in as little as 48 hours in samples incubated at 70°C with +650‰ water. Additionally separate samples that reacted with pure D2O develop a clear infrared signal at ~ 2600 cm-1 due to OD bond stretching. 2) Initially 2 wt% H2O ash is hydrated to nearly 4 wt% of which around 1% is surface correlated water removable by a few days of drying. 3) The loosely bound water is enriched in deuterium by roughly 20‰ over that which remains in the dried native ash. This is inferred by comparing isotopic ratios of dried and un-dried ash and assuming linear mixing. 4) Dried ash without the surface water evinces a much higher rate of deuterium incorporation when compared to rates seen with the un-dried material under all the same conditions. This suggests the less strongly bound water serves as a buffer mediating the relationship between water in the environment and the inner OH more strongly held within the Mt. Mazama ash. We hypothesize that where this surface correlated water is present the movement of deuterium by hydrogen-deuterium exchange or diffusion has a rate determining effect. Where the surface-bound water is absent the rate of deuterium incorporation apparently follows a different mechanism. It appears that deuterium uptake is dominated

  11. Methylamine-hydrogen exchange. Part I - theoretical evaluation of separation factors

    International Nuclear Information System (INIS)

    Dave, S.M.; Ghosh, S.K.; Sadhukhan, H.K.

    1981-01-01

    The equilibrium constants and the deuterium separation factors for the methylamine-hydrogen exchange when all the species of these molecules are at equilibrium, have been calculated as function of temperature and deuterium concentrations in amine and hydrogen, using recent spectroscopic data. Wide range of temperature and deuterium concentrations have been covered. It has been observed that the separation factors for this system are higher than the corresponding fractionation factors for ammonia-hydrogen exchange. From the temperature dependence of these separation factors, the heat of reaction for the isotopic exchange between amine and hydrogen has been calculated. (author)

  12. Efficiency of Al2O3 supported palladium sorbents in the process of hydrogen isotope exchange

    International Nuclear Information System (INIS)

    Andreev, B.M.; Perevezentsev, A.N.; Yasenkov, V.I.

    1981-01-01

    It is found that in the hydrogen-palladium system while applying the metal to aluminium oxide a considerable increase of the heterogeneous hydrogen isotopic exchange rate is observed due to the increase of its specific surface at 167-298 K temperatures and 350-500 Torr hydrogen pressures. It is shown that in the process of thermal treatment of the supported palladium sorbent resulting in reconstruction of the carrier porous structure, as well as in increasing the metal crystal size, the change of the stage, limiting the isotopic exchange process, occurs. The values of the rate and energy of activation of the hydrogen isotopic exchange are presented [ru

  13. Relative extents of hydrogen-deuterium exchange of nitrosamines: relevance to biological isotope effect studies

    International Nuclear Information System (INIS)

    Singer, G.M.; Lijinsky, W.

    1979-01-01

    Relative extents of base-catalyzed, hydrogen-deuterium exchange have been determined for a number of nitrosamines. Observed trends in the exchanges are discussed in terms of substitution, ring size and conformation. The relevance of the exchanges to deuterium isotope effects in carcinogenesis tests is discussed. Those compounds which give pronounced biological isotope effects undergo exchange only to a small extent. No biological isotope effect is found for compounds which undergo extensive exchange. (author)

  14. Hydrogen/deuterium exchange in electrospray ionization mass spectrometry as a practical tool for structure elucidation

    International Nuclear Information System (INIS)

    Conboy, J.J.; Bean, M.F.; Hemling, M.E.; Carr, S.A.

    1993-01-01

    Measurement of the number of active hydrogens in a molecule by various types of hydrogen/deuterium exchange experiments has for a number of years proven useful in the elucidation of compounds of unknown structure, in the determination of the mechanism of chemical or biological reactions, and in the interpretation of fragmentation pathways in mass spectra. Recent work in the analysis of protein conformation by ESIMS has made use of both solution phase and gas phase (atmospheric pressure or in vacuo) hydrogen/deuterium exchange methods. The gas phase exchange experiments were performed either on FTMS instrumentation to achieve the long residence times required for exchange or with a heated dual-capillary inlet reactor interface for desolvation and then exchange of the sample molecules. The authors have implemented methods for hydrogen/deuterium exchange which require only simple changes to the plumbing of a Sciex API-III Ionspray source. Effective exchange of active hydrogens may be achieved by replacing either all of the nebulizer gas or part of the curtain gas or both with a deuterated exchange agent such as ND 3 . These methods provide for essentially complete exchange of all active hydrogens on the types of small molecules (e.g., alkaloids, glycosides, etc.) for which H/D exchange has been of proven utility in structure elucidation work; larger molecules, or at least those with a large number of potentially exchangeable hydrogens, proved to be more challenging particularly in regard to completeness of exchange. These methods also appear to work without the need for dissolving the sample in deuterated or aprotic solvents. A simple ESIMS H/D exchange experiment is a useful addition to the mass spectrometrist's armamentarium for the elucidation of unknown structures. With it, H/D exchange is now extended to classes of compounds not amenable to FAB or DCI, especially in cases where sample is limited

  15. Metal and hydrogen catalysis in isotopic hydrogen exchange in some biologically important heterocyclic compounds

    International Nuclear Information System (INIS)

    Buncel, E.; Joly, H.A.; Jones, J.R.; Onyido, I.

    1989-01-01

    This study reports on the catalytic roles of metal and hydrogen ions in tritium exchange in some heterocyclic substrates which occur as residues in many biologically important molecules. We have found that detritiation of 1-methyl[2- 3 H]imidazole is inhibited by a number of metal ions. As well, inhibition of exchange rates was noted with Ag(I) and Cu(II) for [2- 3 H]thiazole and 1-methyl[8- 3 H]inosine, with Ag(I) for [2- 3 H]benzothiazole, and with Cu(II) for 1-methyl[8- 3 H]guanosine. A complete mechanistic description, which includes the various metal ion-coordinated species generated under the experimental conditions, is presented. The results demonstrate the reactivity order: protonated >> metal-coordinated >> neutral substrates. The differential catalytic effects of metal and hydrogen ions in these processes are discussed in terms of the extent of charge developed on the ligating heteroatom in the reaction intermediate. (author). 13 refs.; 1 fig

  16. Difluoroborane, a hydrogen-deuterium exchange catalyst. Application to pentaborane (9) and methylpentaborane (9)

    International Nuclear Information System (INIS)

    Kline, G.A.; Porter, R.F.

    1981-01-01

    The hydrogen exchange reaction of pentaborane with difluoroborane suggests the existence of a multicentered intermediate similar to that proposed by DeStefano and Porter for the difluoroborane-borazine exchange reaction. A similar intermediate may apply to the borane-pentaborane exchange reaction

  17. Rapid and Controllable Hydrogen/Deuterium Exchange on Aromatic Rings of α-Amino Acids and Peptides

    OpenAIRE

    Murai, Yuta; Wang, Lei; Masuda, Katsuyoshi; Sakihama, Yasuko; Hashidoko, Yasuyuki; Hatanaka, Yasumaru; Hashimoto, Makoto

    2013-01-01

    Novel hydrogen/deuterium exchange for aromatic α-amino acids and their corresponding peptides were performed through the use of deuterated trifluoromethanesulfonic acid (TfOD). Detailed analysis of the exchange revealed that equal hydrogen/deuterium exchange was observed for phenylalanine, and specific exchange at the ortho-positions of phenol for tyrosine was also detected. The stereochemistry of the aromatic α-amino acids was retained under the exchange conditions. The hydrogen/deuterium ex...

  18. Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry.

    Science.gov (United States)

    Yang, Menglin; Hoeppner, Morgan; Rey, Martial; Kadek, Alan; Man, Petr; Schriemer, David C

    2015-07-07

    The pitcher secretions of the Nepenthes genus of carnivorous plants contain a proteolytic activity that is very useful for hydrogen/deuterium exchange mass spectrometry (HX-MS). Our efforts to reconstitute pitcher fluid activity using recombinant nepenthesin I (one of two known aspartic proteases in the fluid) revealed a partial cleavage profile and reduced enzymatic stability in certain HX-MS applications. We produced and characterized recombinant nepenthesin II to determine if it complemented nepenthesin I in HX-MS applications. Nepenthesin II shares many properties with nepenthesin I, such as fast digestion at reduced temperature and pH, and broad cleavage specificity, but in addition, it cleaves C-terminal to tryptophan. Neither enzyme reproduces the C-terminal proline cleavage we observed in the natural extract. Nepenthesin II is considerably more resistant to chemical denaturants and reducing agents than nepenthesin I, and it possesses a stability profile that is similar to that of pepsin. Higher stability combined with the slightly broader cleavage specificity makes nepenthesin II a useful alternative to pepsin and a more complete replacement for pitcher fluid in HX-MS applications.

  19. Hexicon 2: Automated Processing of Hydrogen-Deuterium Exchange Mass Spectrometry Data with Improved Deuteration Distribution Estimation

    Science.gov (United States)

    Lindner, Robert; Lou, Xinghua; Reinstein, Jochen; Shoeman, Robert L.; Hamprecht, Fred A.; Winkler, Andreas

    2014-06-01

    Hydrogen-deuterium exchange (HDX) experiments analyzed by mass spectrometry (MS) provide information about the dynamics and the solvent accessibility of protein backbone amide hydrogen atoms. Continuous improvement of MS instrumentation has contributed to the increasing popularity of this method; however, comprehensive automated data analysis is only beginning to mature. We present Hexicon 2, an automated pipeline for data analysis and visualization based on the previously published program Hexicon (Lou et al. 2010). Hexicon 2 employs the sensitive NITPICK peak detection algorithm of its predecessor in a divide-and-conquer strategy and adds new features, such as chromatogram alignment and improved peptide sequence assignment. The unique feature of deuteration distribution estimation was retained in Hexicon 2 and improved using an iterative deconvolution algorithm that is robust even to noisy data. In addition, Hexicon 2 provides a data browser that facilitates quality control and provides convenient access to common data visualization tasks. Analysis of a benchmark dataset demonstrates superior performance of Hexicon 2 compared with its predecessor in terms of deuteration centroid recovery and deuteration distribution estimation. Hexicon 2 greatly reduces data analysis time compared with manual analysis, whereas the increased number of peptides provides redundant coverage of the entire protein sequence. Hexicon 2 is a standalone application available free of charge under http://hx2.mpimf-heidelberg.mpg.de.

  20. The Area Between Exchange Curves as a Measure of Conformational Differences in Hydrogen-Deuterium Exchange Mass Spectrometry Studies

    Science.gov (United States)

    Mazur, Sharlyn J.; Weber, Daniel P.

    2017-05-01

    Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides information about protein conformational mobility under native conditions. The area between exchange curves, A bec , a functional data analysis concept, was adapted to the interpretation of HDX-MS data and provides a useful measure of exchange curve dissimilarity for tests of significance. Importantly, for most globular proteins under native conditions, A bec values provide an estimate of the log ratio of exchange-competent fractions in the two states, and thus are related to differences in the free energy of microdomain unfolding.

  1. Mechanisms of hydrogen exchange in proteins from nuclear magnetic resonance studies of individual tryptophan indole NH hydrogens in lysozyme

    International Nuclear Information System (INIS)

    Wedin, R.E.; Delepierre, M.; Dobson, C.M.; Poulsen, F.M.

    1982-01-01

    The individual rates of solvent exchange of the six tryptophan indole NH hydrogens of lysozyme in 2 H 2 O have been measured over a wide range of temperatures by using 1 H NMR. Two distinct mechanisms for exchange have been identified, one characterized by a high activation energy and the other by a much lower activation energy. The high-energy process has been shown to be associated directly with the cooperative thermal unfolding of the protein and is the dominant mechanism for exchange of the most slowly exchanging hydrogen even 15 0 C below the denaturation temperature. Rate constants and activation energies for the folding and unfolding reactions were obtained from the experimental exchange rates. At low temperatures, a lower activation energy mechanism is dominant for all hydrogens, and this can be associated with local fluctuations in the protein structure which allow access of solvent. The relative exchange rates and activation energies can only qualitatively be related to the different environments of the residues in the crystal structure. There is provisional evidence that a mechanism intermediate between these two extremes may be significant for some hydrogens under restricted conditions

  2. Influence of Murchison or Allende minerals on hydrogen-deuterium exchange of amino acids

    Science.gov (United States)

    Lerner, N. R.

    1995-04-01

    Deuterium-enriched amino acids occur in the Murchison carbonaceous chrondrite. This meteorite underwent a period of aqueous alteration with isotopically light water. With the objective of setting limits on the conditions of aqueous alteration, the exchange of the carbon-bonded hydrogen atoms of amino acids with D 2O has been studied from 295 to 380 K as a function of time and meteorite/heavy water ratio. The amount of Murchison or Allende dust present has a significant effect on the rate and amount of hydrogen-deuterium exchange observed. At elevated temperatures, the a-hydrogens of all the amino acids studied were found to exchange with deuterium. In glycine and aspartic acid, this process resulted in total exchange of the carbon-bonded hydrogen. A completely deuterated isotopomer of alanine was produced in significant quantities only when the rock/water ratio was greater than 0.5. No exchange of carbonbonded hydrogens was observed in the case of amino acids which do not possess an α-hydrogen atom. The rates of H/D exchange for amino acids observed here did not correspond to deuterium enrichment of the amino acids in the Murchison meteorite. These results suggest that H/D exchange with water had a negligible effect on the observed deuterium enrichment of amino acids found in Murchison and that the temperature at which the amino acids were exposed to liquid water was close to 273 K.

  3. Hydrogen-deuterium exchange in bulk LiBH4

    NARCIS (Netherlands)

    Borgschulte, A.; Zuttel, A.; Hug, P.; Racu, A. M.; Schoenes, J.

    2008-01-01

    Because of its apparent simplicity, diffusion of hydrogen in solids can be regarded as a general model system for diffusion. However, only rudimentary knowledge exists for the dynamics of hydrogen in complex hydrides. Insight into the specific diffusion process is given by hydrogen-deuterium

  4. Exchange amplitudes for electron-hydrogen scattering in the Glauber approximation

    International Nuclear Information System (INIS)

    Khayrallah, G.

    1976-01-01

    An exact closed form expression for the Glauber-Bonham-Ochkur exchange amplitudes for electron-hydrogen scattering is derived. The results are applied to the elastic scattering from the ground state of the hydrogen atom, where a closed-form expression was also derived for the total exchange cross section. Investigation of the inclusion of exchange in the differential cross section for total electron scattering is also presented. It is shown that exchange effects are quite important and that their inclusion, rather than the inclusion of angle effect or the inclusion of the full eikonal effects, does tend to make the Glauber prediction in much better agreement with the experiment

  5. The effect of glucosyl-β-cyclodextrin on the hydrogen-deuterium exchange rate constant of the peptide bonds of chicken egg white lysozyme in a D 2O solution

    Science.gov (United States)

    Yoshikiyo, Keisuke; Sugimoto, Masatoshi; Aso, Yuji; Takahashi, Tetsuya; Matsui, Yoshihisa; Yamamoto, Tatsuyuki

    2008-10-01

    FT-IR spectroscopy revealed that the hydrogen-deuterium (H-D) exchange reaction rate of the peptide hydrogen atoms of chicken egg white lysozyme in a deuterated aqueous solution was significantly accelerated in the presence of glucosyl-β-cyclodextrin at 55 °C. The addition of methyl α- D-glucopyranoside, which has no inclusion ability, rather decelerated the H-D exchange reaction rate at the same temperature. The H-D exchange rate constant of lysozyme was evaluated by the time dependence of the absorbance ratio of the amide II infrared band against the amide I'. The H-D exchange rate constant was not influenced by the addition of glucosyl-β-cyclodextrin at 45 °C, however, it became twice larger than that in the absence of the cyclodextrin at 55 °C. These results strongly suggest that peptide bonds of lysozyme become exposed to the aqueous medium due to the inclusion by glucosyl-β-cyclodextrin to accelerate the H-D exchange rate.

  6. Sodium hydrogen exchangers contribute to arenavirus cell entry.

    Science.gov (United States)

    Iwasaki, Masaharu; Ngo, Nhi; de la Torre, Juan C

    2014-01-01

    Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a great public health concern in the regions in which they are endemic. Moreover, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The limited existing armamentarium to combat human-pathogenic arenaviruses underscores the importance of developing novel antiarenaviral drugs, a task that would be facilitated by the identification and characterization of virus-host cell factor interactions that contribute to the arenavirus life cycle. A genome-wide small interfering RNA (siRNA) screen identified sodium hydrogen exchanger 3 (NHE3) as required for efficient multiplication of LCMV in HeLa cells, but the mechanisms by which NHE activity contributed to the life cycle of LCMV remain unknown. Here we show that treatment with the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) resulted in a robust inhibition of LCMV multiplication in both rodent (BHK-21) and human (A549) cells. EIPA-mediated inhibition was due not to interference with virus RNA replication, gene expression, or budding but rather to a blockade of virus cell entry. EIPA also inhibited cell entry mediated by the glycoproteins of the HF arenaviruses LASV and Junin virus (JUNV). Pharmacological and genetic studies revealed that cell entry of LCMV in A549 cells depended on actin remodeling and Pak1, suggesting a macropinocytosis-like cell entry pathway. Finally, zoniporide, an NHE inhibitor being explored as a therapeutic agent to treat myocardial infarction, inhibited LCMV propagation in culture cells. Our findings indicate that targeting NHEs could be a novel strategy to combat human-pathogenic arenaviruses.

  7. Regio-controlled hydrogen-deuterium exchange of biologically important indoles under uv irradiation

    International Nuclear Information System (INIS)

    Saito, Isao; Muramatsu, Shigeru; Sugiyama, Hiroshi; Yamamoto, Akihiro; Matsuura, Teruo

    1985-01-01

    Photochemical hydrogen-deuterium exchange reaction of biologically important indoles is reported. The regioselectivity of the photodeuteration was found to be controlled by the ammonium group of the side chain. (author)

  8. Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

    International Nuclear Information System (INIS)

    Tuechsen, E.; Hayes, J.M.; Ramaprasad, S.; Copie, V.; Woodward, C.

    1987-01-01

    Solvent exchange of 18 O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectroscopy. Buried water is labeled by equilibrium of the protein in 18 O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18 O label eluting with the protein in 10-300 s is determined by an H 2 O-CO 2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH ≥ 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18 O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18 O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18 O labeling of carboxyl groups at low pH. In addition to 18 O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent and hydrogen-bonded main chain O, and their pH/sub min/ is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate

  9. SAIDE: A Semi-Automated Interface for Hydrogen/Deuterium Exchange Mass Spectrometry

    OpenAIRE

    Villar, Maria T.; Miller, Danny E.; Fenton, Aron W.; Artigues, Antonio

    2010-01-01

    Deuterium/hydrogen exchange in combination with mass spectrometry (DH MS) is a sensitive technique for detection of changes in protein conformation and dynamics. Since temperature, pH and timing control are the key elements for reliable and efficient measurement of hydrogen/deuterium content in proteins and peptides, we have developed a small, semiautomatic interface for deuterium exchange that interfaces the HPLC pumps with a mass spectrometer. This interface is relatively inexpensive to bui...

  10. Separation of rate processes for isotopic exchange between hydrogen and liquid water in packed columns 10

    International Nuclear Information System (INIS)

    Butler, J.P.; Hartog, J. den; Goodale, J.W.; Rolston, J.H.

    1977-01-01

    Wetproofed platinum catalysts in packed columns promote isotopic exchange between counter-current streams of hydrogen saturated with water vapour and liquid water. The net rate of deuterium transfer from isotopically enriched hydrogen has been measured and separated into two rate processes involving the transfer of deuterium from hydrogen to water vapour and from water vapour to liquid. These are compared with independent measurements of the two rate processes to test the two-step successive exchange model for trickle bed reactors. The separated transfer rates are independent of bed height and characterize the deuterium concentrations of each stream along the length of the bed. The dependences of the transfer rates upon hydrogen and liquid flow, hydrogen pressure, platinum loading and the effect of dilution of the hydrophobic catalyst with inert hydrophilic packing are reported. The results indicate a third process may be important in the transfer of deuterium between hydrogen and liquid water. (author)

  11. Photochemically induced hydrogen isotope exchange between anthracene and aliphatic amines

    International Nuclear Information System (INIS)

    Gebicki, J.; Reimschuessel, W.; Nowicki, T.

    1978-01-01

    The kinetics of photochemically induced deuterium and tritium isotope exchange between anthracene and diethylamine labelled in NH group or triethylamine-labelled water mixture was studied in acetonitrile and n-heptane. The rate constants of isotope exchange were calculated and ksub(D)/ksub(T) isotope effect was found. A mechanism for isotope exchange involving exciplex formation was proposed. (Auth.)

  12. Determination of limiting stage of interface hydrogen isotopic exchange on NaX-3M zeolite

    International Nuclear Information System (INIS)

    Polevoj, A.S.; Yudin, I.P.

    1987-01-01

    The dependence of transfer unit height, when separating the mixture H 2 -D 2 using NaX-3M zeolite, on the value of gas consumption and sorbent grain size in a countercurrent column is determined. Hydrogen diffusion in sorbent secondary pores is shown to be the limiting stage of interface hydrogen isotopic exchange on NaX-3M zeolite

  13. Online hydrogen/deuterium exchange performed in the ion mobility cell of a hybrid mass spectrometer.

    Science.gov (United States)

    Nagy, Kornél; Redeuil, Karine; Rezzi, Serge

    2009-11-15

    The present paper describes the performance of online, gas-phase hydrogen/deuterium exchange implemented in the ion mobility cell of a quadrupole time-of-flight mass spectrometer. Deuterium oxide and deuterated methanol were utilized to create deuterated vapor that is introduced into the ion mobility region of the mass spectrometer. Hydrogen/deuterium exchange occurs spontaneously in the milliseconds time frame without the need of switching the instrument into ion mobility mode. The exchange was studied in case of low molecular weight molecules and proteins. The observed number of exchanged hydrogens was equal to the number of theoretically exchangeable hydrogens for all low molecular weight compounds. This method needs only minimal instrumental modifications, is simple, cheap, environment friendly, compatible with ultraperformance liquid chromatography, and can be implemented on commercially available instruments. It does not compromise choice of liquid chromatographic solvents and accurate mass or parallel-fragmentation (MS(E)) methods. The performance of this method was compared to that of conventional alternatives where the deuterated solvent is introduced into the cone gas of the instrument. Although the degree of exchange was similar between the two methods, the "cone gas method" requires 10 times higher deuterated solvent volumes (50 muL/min) and offers reduced sensitivity in the tandem mass spectrometry (MS/MS) mode. The presented method is suggested as a standard future element of mass spectrometers to aid online structural characterization of unknowns and to study conformational changes of proteins with hydrogen/deuterium exchange.

  14. On the proton exchange contribution to electron-hydrogen atom elastic scattering

    International Nuclear Information System (INIS)

    Mignaco, J.A.; Tort, A.C.

    1979-05-01

    It is shown that the exchange contribution to the electron-proton potential Born term in elastic electron-hydrogen atom scattering arises as the non relativistic limit from the exchange of a proton between the two participant electrons - calculated from quantum electrodynamics including properly bound states (as solution of Bethe - Salpeter equation). (Author) [pt

  15. Deuterium separation factors for isotopic exchange between hydrogen sulphide and water

    International Nuclear Information System (INIS)

    Dave, S.M.; Ghosh, S.K.; Sadhukhan, H.K.

    1981-01-01

    The overall deuterium separation factors for the hydrogen sulphide-water exchange where all the isotopic species of these molecules are in equilibrium have been calculated both for liquid-gas and gas-gas exchange as functions of temperature and deuterium concentration. (author)

  16. Hydrogen exchange in hydrated films of proteins. Application to the E. coli lac repressor core

    International Nuclear Information System (INIS)

    Pilet, J.; Szabo, A.G.; Maurizot, J.-C.

    1980-01-01

    An original easy method of hydrogen to deuterium exchange in hydrated films of proteins, followed by infrared absorption measurements, is described and applied to films of the E. coli lac repressor core, in order to examine the effect of isopropyl-β-D-thiogalactoside (IPTG) binding. An estimation of about 25% α helical structure in this protein fragment is deduced from the exchange curve. The binding of IPTG to the core does not affect the exchange curve within the experimental error limits. (Auth.)

  17. Dissecting the mechanism of Epac activation via hydrogen-deuterium exchange FT-IR and structural modeling.

    Science.gov (United States)

    Yu, Shaoning; Fan, Fenghui; Flores, Samuel C; Mei, Fang; Cheng, Xiaodong

    2006-12-26

    Exchange proteins directly activated by cAMP (Epac) make up a family of cAMP binding domain-containing proteins that play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Ras-proximate proteins. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and structural modeling. Our studies show that unlike that of cAMP-dependent protein kinase (PKA), the classic intracellular cAMP receptor, binding of cAMP to Epac does not induce significant changes in overall secondary structure and structural dynamics, as measured by FT-IR and the rate of H-D exchange, respectively. These results suggest that Epac activation does not involve significant changes in the amount of exposed surface areas as in the case of PKA activation, and conformational changes induced by cAMP in Epac are most likely confined to small local regions. Homology modeling and comparative structural analyses of the CBDs of Epac and PKA lead us to propose a model of Epac activation. On the basis of our model, Epac activation by cAMP employs the same underlying structural principal utilized by PKA, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different. In addition, we predict that during Epac activation the first beta-strand of the switchboard switches its conformation to a alpha-helix, which folds back to the beta-barrel core of the CBD and interacts directly with cAMP to form the base of the cAMP-binding pocket.

  18. Deuterium removal from radiation damage in tungsten by isotopic exchange with hydrogen atomic beam

    Science.gov (United States)

    Ogorodnikova, O. V.; Markelj, S.; Efimov, V. S.; Gasparyan, Yu M.

    2016-09-01

    The tungsten samples were pre-irradiated with self-ions to create radiation-induced defects and then exposed to the deuterium atomic beam. The deuterium removal was studied by isotopic exchange with atomic hydrogen beam. Modification of the deuterium depth profile in self-ion irradiated tungsten under isotopic exchange up to a depth of 6 μm was measured in- situ by nuclear reaction analysis. The total deuterium retention after isotopic exchange was measured by thermal desorption spectroscopy. It is shown that the efficiency of the deuterium removal increases with increasing of the hydrogen incident flux, incident energy and temperature of the tungsten sample.

  19. On the mechanism of hydrogen-deuterium exchange in bacteriorhodopsin.

    OpenAIRE

    Doukas, A G; Pande, A; Suzuki, T; Callender, R H; Honig, B; Ottolenghi, M

    1981-01-01

    Continuous-flow resonance Raman experiments carried out in bacteriorhodopsin show that the exchange of a deuteron on the Schiff base with a proton takes place in times shorter than 3 ms. Exchange mechanisms based on a base-catalyzed deprotonation followed by reprotonation of the Schiff base are excluded. A mechanism is suggested in which a water molecule interacts directly with the Schiff base deuteron in a concerted exchange mechanism. It appears that in the dark, the binding site is more ac...

  20. Structure and intermolecular dynamics of aggregates populated during amyloid fibril formation studied by hydrogen/deuterium exchange.

    Science.gov (United States)

    Carulla, Natàlia; Zhou, Min; Giralt, Ernest; Robinson, Carol V; Dobson, Christopher M

    2010-08-17

    The aggregation of proteins into amyloid fibrils is a complex and fascinating process associated with debilitating clinical disorders such as Alzheimer's and Parkinson's diseases. The process of aggregation involves a series of steps during which many intermediate aggregation states are populated. Recent evidence points to these intermediate states as the toxic moieties primarily responsible for cell damage or cell death, which are critical steps in the origin and progression of these disorders. To understand the molecular basis of these diseases, it is crucial to investigate and define the details of the aggregation process, and to achieve this objective, researchers need the tools to characterize the structure and kinetics of interconversion of the various species present during amyloid fibril formation. Hydrogen-deuterium (HD) exchange experiments are based on solvent accessibilities and provide one means by which this kind of information may be acquired. In this Account, we describe research based on HD exchange processes that is directed toward better understanding the dynamics and structural reorganizations involved in the formation of amyloid fibrils. Amide hydrogens that normally undergo rapid exchange with solvent hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent. The rates of exchange can be monitored by replacing some hydrogens with deuterons. When peptide and protein molecules assemble into amyloid fibrils, the fibrils contain a core region based on repetitive arrays of beta-sheets oriented parallel to the fibril axis. HD experiments have been applied extensively to map such structures in different amyloid fibril systems. By an extension of this approach, we have observed that HD exchange can be governed by a mechanism through which molecules making up the fibrils are continuously dissolving and reforming, revealing that amyloid fibrils are not static but dynamic structures

  1. Analysis of conformational changes in rhodopsin by histidine hydrogen-deuterium exchange.

    Science.gov (United States)

    Lodowski, David T; Miyagi, Masaru

    2015-01-01

    Hydrogen-deuterium exchange (HDX) is a technique that measures the exchange of protein hydrogens for deuteriums in a D2O-containing buffer, providing readout of the structural dynamics. Histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) is a variation of this technique that measures the slow HDX of imidazole C2 hydrogens of histidines. This measurement, when accompanied by pH titration, provides both pK as and half-lives (t 1/2) of the HDX reaction for individual histidine residues in proteins. The pK a and t 1/2 values indicate the electrostatic environment and the degree of side-chain solvent accessibility of the histidine residues, respectively. Herein we describe an experimental protocol to characterize rhodopsin by His-HDX-MS. This technique can be used to monitor different states of rhodopsin and might be useful for monitoring longtime scale events in other GPCRs.

  2. Hydrogen-Deuterium Exchange in Photolyzed Methane-Water Ices

    Science.gov (United States)

    Weber, Amanda S.; Hodyss, Robert; Johnson, Paul V.; Willacy, Karen; Kanik, Isik

    2009-09-01

    Previous work has concluded that H-D exchange occurs readily in polycyclic aromatic hydrocarbons frozen in deuterated water (D2O) irradiated with ultraviolet light. Here, we examine H-D exchange in methane-water ices following exposure to ultraviolet radiation and analyze the products formed as a result. We find that H-D exchange also occurs in methane-water ices by means of ultraviolet photolysis. Exchange proceeds through a radical mechanism that implies that almost all organic species will undergo significant H-D exchange with the matrix in water ices exposed to ultraviolet radiation. Given sufficient energetic processing of the ice, the H/D ratio of an ice matrix may be transferred to the organic species in the ice.

  3. HYDROGEN-DEUTERIUM EXCHANGE IN PHOTOLYZED METHANE-WATER ICES

    International Nuclear Information System (INIS)

    Weber, Amanda S.; Hodyss, Robert; Johnson, Paul V.; Willacy, Karen; Kanik, Isik

    2009-01-01

    Previous work has concluded that H-D exchange occurs readily in polycyclic aromatic hydrocarbons frozen in deuterated water (D 2 O) irradiated with ultraviolet light. Here, we examine H-D exchange in methane-water ices following exposure to ultraviolet radiation and analyze the products formed as a result. We find that H-D exchange also occurs in methane-water ices by means of ultraviolet photolysis. Exchange proceeds through a radical mechanism that implies that almost all organic species will undergo significant H-D exchange with the matrix in water ices exposed to ultraviolet radiation. Given sufficient energetic processing of the ice, the H/D ratio of an ice matrix may be transferred to the organic species in the ice.

  4. A Two-Stage Differential Hydrogen Deuterium Exchange Method for the Rapid Characterization of Protein/Ligand Interactions

    Science.gov (United States)

    Chalmers, Michael J.; Busby, Scott A.; Pascal, Bruce D.; Southern, Mark R.; Griffin, Patrick R.

    2007-01-01

    The peroxisome proliferator-activated receptor is a member of the nuclear receptor superfamily of transcriptional regulators. Regulation of the nuclear receptors occurs through changes to the structure and dynamics of the ligand-binding domain. Therefore, the need has arisen for a rapid method capable of detecting changes in the dynamics of nuclear receptors following ligand binding. We recently described how solution-phase amide hydrogen/deuterium exchange (HDX) provides a biophysical technique for probing changes in protein dynamics induced by ligand interaction. Building from this platform, we have optimized the robustness of the differential HDX experiment by minimizing systematic errors, and have increased the efficiency of the chromatographic separation through the use of high-pressure liquid chromatography. Using knowledge gained previously from comprehensive HDX experiments of PPARγ, a modest throughput method to probe changes in the dynamics of key regions of the receptor was developed. A collection of ten synthetic and endogenous PPARγ ligands were characterized with this new method requiring approximately 24 h of analysis. This is a dramatic improvement over the 10 d of analysis that would have been required with our previous approach for comprehensive differential HDX analysis. In addition to demonstrating the utility of this approach, the study presented here is the first to measure changes to the dynamics of PPARγ upon the binding of putative endogenous ligands. PMID:17916792

  5. Contribution to the study of isotopic exchange of hydrogen in purine derivatives

    International Nuclear Information System (INIS)

    Valenta, V.

    1977-01-01

    The dependence was studied of the rate of hydrogen isotopic exchange in purine derivatives in aqueous media. The effect of pH and of temperature was investigated in adenosine, adenosine monophosphate and adenosine triphosphate, and of pH in guanosine, guanosine monophosphate and guanosine triphosphate. The isotopic exchange of hydrogen was studied in the system hexamethyl phosphotriamide - [ 3 H]water - purine derivative (adenine, adenosine, adenosine monophosphate). The reaction mechanism of hydrogen isotopic exchange is discussed. It may be inferred from the measured value of the kinetic isotopic effect. The ''reverse'' isotopic exchange of hydrogen was investigated in [8- 3 H]adenosine, [8- 3 H]adenosine-5'-monophosphate, and [8- 3 H]adenosine-5'-triphosphate. The possible use was verified of the hydrogen isotopic exchange in the system tritium gas - water - purine derivative, catalyzed by metal catalysts, for the preparation of purine derivatives labelled with tritium in position 8. The effect of pH of, the catalyst type and of quantity was studied. Adenosine-5'-triphosphate with a molar activity of 860 GBq/mmol was prepared using carrier-free tritium gas. (author)

  6. Hydrogen exchange at the β-carbon of amino acids during transamination

    International Nuclear Information System (INIS)

    Walter, U.; Luthe, H.; Soeling, H.D.; Gerhart, F.

    1975-01-01

    The hydrogen exchange at the β-carbon of L-alanine, L-glutamate and L-aspartate with water has been examined during transamination catalyzed by glutamic-oxaloacetic transaminase and by glutamic-pyruvic transaminase. A significant hydrogen exchange at the β-carbon has been demonstrated during incubation of L-[3- 3 H] alanine + glutamic-pyruvic transaminase, L-[3- 3 H] alanine + α-oxoglutarate + glutamic-pyruvic transaminase, L-[3- 3 H] glutamate + glutamic-oxaloacetic transaminase, L-[3- 3 H] glutamate + oxalocetate + glutamic-oxaloacetic transaminase, and L-[3- 3 H] glutamate + pyruvate + glutamic-pyruvic transaminase as shown by the appearance of 3 H 2 O. No hydrogen exchange at the β-carbon of L-glutamate occurred during incubation of L-[3- 3 H] -glutamate with glutamic-pyruvic transaminase alone. The hydogen exchange at the β-carbon of L-glutamate coincides with transamination as demonstrated by nuclear magnetic resonance studies of 2 H 2 O-L-glutamate exchange during transamination by glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase. No hydrogen exchange at the β-carbon occurred during transamination of L-aspartate by glutamic-oxaloacetic transaminase as shown by nuclear magnetic resonance spectroscopy. The results are discussed with special reference to the different equilibria between the pyridoxal form and the pyridoxamine form of glutamic-oxaloacetic transaminase and of glutamic-pyruvic transaminase. (orig.) [de

  7. Activation of molecular hydrogen by solid and molten hydroxides. III. Kinetics and mechanism of isotope exchange between hydrogen and solutions of water in alkali melts

    International Nuclear Information System (INIS)

    Baikov, Yu.M.; Ryskin, G.Ya.

    1982-01-01

    A kinetic study has been made of deuterium-protium exchange between hydrogen and water/caustic mixtures containing up to 50% (mole) water. It has been shown that the reaction proceeds homogeneously in the bulk liquid phase with the participation of dissolved hydrogen molecules. It is suggested that the associate OH - x H 2 O participates in the limiting stage of the exchange

  8. 1H NMR studies of substrate hydrogen exchange reactions catalyzed by L-methionine gamma-lyase

    International Nuclear Information System (INIS)

    Esaki, N.; Nakayama, T.; Sawada, S.; Tanaka, H.; Soda, K.

    1985-01-01

    Hydrogen exchange reactions of various L-amino acids catalyzed by L-methionine gamma-lyase (EC 4.4.1.11) have been studied. The enzyme catalyzes the rapid exchange of the alpha- and beta-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium from the solvent. The rate of alpha-hydrogen exchange was about 40 times faster than that of the enzymatic elimination reaction of the sulfur-containing amino acids. The enzyme also catalyzes the exchange reaction of alpha- and beta-hydrogens of the straight-chain L-amino acids which are not susceptible to elimination. The exchange rates of the alpha-hydrogen and the total beta-hydrogens of L-alanine and L-alpha-aminobutyrate with deuterium followed first-order kinetics. For L-norvaline, L-norleucine, S-methyl-L-cysteine, and L-methionine, the rate of alpha-hydrogen exchange followed first-order kinetics, but the rate of total beta-hydrogen exchange decreased due to a primary isotope effect at the alpha-position. L-Phenylalanine and L-tryptophan slowly underwent alpha-hydrogen exchange. The pro-R hydrogen of glycine was deuterated stereospecifically

  9. System for exchange of hydrogen between liquid and solid phases

    Science.gov (United States)

    Reilly, J.J.; Grohse, E.W.; Johnson, J.R.; Winsche, W.E.

    1985-02-22

    The reversible reaction M + x/2 H/sub 2/ reversible MH/sub x/, wherein M is a reversible metal hydride former that forms a hydride MH/sub x/ in the presence of H/sub 2/, generally used to store and recall H/sub 2/, is found to proceed under an inert liquid, thereby reducing contamination, providing better temperature control, providing in situ mobility of the reactants, and increasing flexibility in process design. Thus, a slurry of particles of a metal hydride former with an inert solvent is subjected to a temperature and pressure controlled atmosphere containing H/sub 2/, to store hydrogen and to release previously stored hydrogen. The direction of the flow of the H/sub 2/ through the liquid is dependent upon the H/sub 2/ pressure in the gas phase at a given temperature. When the actual H/sub 2/ pressure is above the equilibrium absorption pressure of the respective hydride the reaction proceeds to the right, i.e., the metal hydride is formed and hydrogen is stored in the solid particle. When the actual pressure in the gas phase is below the equilibrium dissociation pressure of the respective hydride the reaction proceeds to the left, the metal hydride is decomposed and hydrogen is released into the gas phase.

  10. Conformational dynamics of free and catalytically active thermolysin are indistinguishable by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Liu, Yu-Hong; Konermann, Lars

    2008-06-17

    Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.

  11. Concerning the interception of atoms in the radiochemical exchange reaction between hydrogen and deuterium

    International Nuclear Information System (INIS)

    Mund, W.; Uyskens, P.; Lories, R.

    Equations are derived which express the probability of capture of a H or D atom, the total ionic yields in atom-molecule collisions, and the inhibiting effects of different molecules on atomic capture. The inhibiting characteristics of CCl 4 and CCl 3 H in deuterium exchange reactions determined experimentally were sufficiently weak to cause abandonment of the hypothesis that a hydrogen atom in a molecule might be the site of a hydrogen bridge

  12. Anomalous hydrogen-deuterium exchange of cyclic β-keto sulfides

    International Nuclear Information System (INIS)

    Guth, J.J.; Gross, R.L.; Carson, F.W.

    1982-01-01

    The protons at carbon-4 display a higher kinetic acidity than those at carbon-2 in thiolanone when the hydrogen deuterium exchange is catalyzed by pyridine. The purpose of this study was to measure and compare the rates of hydrogen-deuterium exchange of thiolanone and three other ketones 3-pentanone, cyclopentanone, and 1-(methylthio)-2-propanone. Results are reported using 3-pentanone as a standard. They demonstrate that the kinetic acidity of C-H bonds at carbon 4 of thiolanone is approx. 1000 to 5000 times higher than those at carbon 2 and carbon 4 of 3-pentanone

  13. New method for the hydrogen isotope exchange reaction in a hydrophobic catalyst bed

    International Nuclear Information System (INIS)

    Asakura, Y.; Kikuchi, M.; Yusa, H.

    1982-01-01

    To improve the isotope exchange reaction efficiency between water and hydrogen, a new reactor in which water mists and hydrogen gas react cocurrently was studied. To apply this to the enrichment of tritium in heavy water, a dual temperature isotope exchange reactor which is composed of cocurrent low temperature reactors and the usual countercurrent high temperature reactor was proposed and analyzed using a McCabe-Thiele diagram. By utilizing cocurrent reactors, in combination, the necessary catalyst volume can be reduced to one-tenth as compared with the usual countercurrent low temperature reactor. 17 refs

  14. A new type separation column for the water-hydrogen isotope catalytic exchange process

    International Nuclear Information System (INIS)

    Fedorchenko, O.A.; Alekseev, I.A.; Trenin, V.D.

    2001-01-01

    The catalytic water/hydrogen isotope exchange process is by right considered the most attractive for the solution a number of urgent problems of hydrogen isotope separation. A new type exchange reaction column is described and studied in details by computer simulation and with the help of McCabe-Thiele diagrams. It is shown that the new column in comparison with a traditional one needs less catalyst quantity and a smaller diameter for the solving of the same separation tasks. Generalized calculation data are presented in graphical form

  15. A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease*

    Science.gov (United States)

    Englander, S. Walter

    2012-01-01

    A new method for measuring the hydrogen exchange of macromolecules in solution is described. The method uses tritium to trace the movement of hydrogen, and utilizes Sephadex columns to effect, in about 2 minutes, a separation between tritiated macromolecule and tritiated solvent great enough to allow the measurement of bound tritium. High sensitivity and freedom from artifact is demonstrated and the possible value of the technique for investigation of other kinds of colloid-small molecule interaction is indicated. Competition experiments involving tritium, hydrogen, and deuterium indicate the absence of any equilibrium isotope effect in the ribonuclease-hydrogen isotope system, though a secondary kinetic isotope effect is apparent when ribonuclease is largely deuterated. Ribonuclease shows four clearly distinguishable kinetic classes of exchangeable hydrogens. Evidence is marshaled to suggest the independently measurable classes II, III, and IV (in order of decreasing rate of exchange) to represent “random-chain” peptides, peptides involved in α-helix, and otherwise shielded side-chain and peptide hydrogens, respectively. PMID:14075117

  16. Regioselective hydrogen isotope exchange reaction in benzoic acid and its alkali metal salts

    International Nuclear Information System (INIS)

    Nakagawa, Akiko; Hasegawa, Hideaki; Oohashi, Kunio; Seki, Hiroko.

    1997-01-01

    The hydrogen isotope exchange reaction of benzoic and acid its alkali metal salts with deuterium oxide was studied in the presence of RhCl 3 ·3H 2 O. The products were analyzed by 1 H- and 13 C-NMR spectroscopies. High regioselectivity of the exchange at the ortho positions was established, and the extent of deuterium labeling and the distribution of d 0 , d 1 , and d 2 were determined. The reaction mechanism was briefly discussed. (author)

  17. Hydrogen isotope exchange in a metal hydride tube

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, David B. [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

    2014-09-01

    This report describes a model of the displacement of one hydrogen isotope within a metal hydride tube by a different isotope in the gas phase that is blown through the tube. The model incorporates only the most basic parameters to make a clear connection to the theory of open-tube gas chromatography, and to provide a simple description of how the behavior of the system scales with controllable parameters such as gas velocity and tube radius. A single tube can be seen as a building block for more complex architectures that provide higher molar flow rates or other advanced design goals.

  18. Radiation-related retrograde hydrogen isotope and K-Ar exchange in clay minerals

    International Nuclear Information System (INIS)

    Halter, C.; Pagel, M.; Sheppard, S.M.F.; Weber, F.; Clauer, N.

    1987-01-01

    Hydrogen and oxygen isotope studies have been widely applied to characterize the origin of fluids during ore-foaming processes. The primary isotope record, however, may be disturbed by retrograde exchange reactions, thus complicating the interpretation of the data. The susceptibility of minerals to retrograde isotope and chemical exchange is variable, reflecting differences in the mechanism and rate of isotope exchange. Results are presented on deuterium depletion, K/Ar ages and H 2 O + content of illites associated with uranium mineralization from the Athabasca basin (Canada). (author)

  19. Ion exchange reactions of n-butylamine intercalates of tin(IV) hydrogen phosphate and hydrogen uranyl phosphate with cobalt(III) complexes

    International Nuclear Information System (INIS)

    Pozas-Tormo, R.; Moreno-Real, L.; Martinez-Lara, M.; Rodriguez-Castellon, E.

    1986-01-01

    The ion exchange reactions of n-butylamine intercalates of tin(IV) hydrogen phosphate and hydrogen uranyl phosphate towards carbonatotetraamminecobalt(III), chloropentaamminecobalt(III), and hexamminecobalt(III) have been investigated. Independent of the complex cation charges, the amounts of Co(III) complex exchanged by the n-butylamine intercalate of tin(IV) hydrogen phosphate are practically the same. With the n-butylamine intercalate of hydrogen uranyl phosphate, the ionic exchange was completed and the composition was fixed by the exchanged Co(III) complex. The layer charge densities of these phosphates justify the different ionic exchange behaviour observed towards the large complex cations. All the products were characterized by chemical analysis, X-ray diffractory, infrared spectroscopy, diffuse reflectance spectroscopy, and thermal analysis

  20. Hyperfine contributions to spin-exchange frequency shifts in the hydrogen maser

    NARCIS (Netherlands)

    Verhaar, B.J.; Koelman, J.M.V.A.; Stoof, H.T.C.; Luiten, O.J.; Crampton, S.B.

    1987-01-01

    We have rigorously included hyperfine interactions during electron-spin-exchange collisions between ground state hydrogen atoms. We predict additional frequency shifts which are not compensated for by the usual methods of tuning maser cavities. These shifts are large compared to the potential

  1. Dual Studies on a Hydrogen-Deuterium Exchange of Resorcinol and the Subsequent Kinetic Isotope Effect

    Science.gov (United States)

    Giles, Richard; Kim, Iris; Chao, Weyjuin Eric; Moore, Jennifer; Jung, Kyung Woon

    2014-01-01

    An efficient laboratory experiment has been developed for undergraduate students to conduct hydrogen-deuterium (H-D) exchange of resorcinol by electrophilic aromatic substitution using D[subscript 2]O and a catalytic amount of H[subscript 2]SO[subscript 4]. The resulting labeled product is characterized by [superscript 1]H NMR. Students also…

  2. Hydrogen/deuterium exchange of phenylalanine analogs studied with infrared multiple photon dissociation

    NARCIS (Netherlands)

    Contreras, C. S.; Polfer, N. C.; Chung, A. C.; Oomens, J.; Eyler, J. R.

    2010-01-01

    Phenylalanine analogs were subjected to hydrogen/deuterium exchange (HDX) in both solution and the gas phase, and gas-phase infrared multiple photon dissociation spectra were obtained for each of the species. For sodium cation-attached N-acetylphenylalanine, gas-phase HDX took place at only one

  3. Deuterium labelled lipophylic steroid analogues. Useful hydrogen-deuterium exchange in the course of Wittig reaction

    Czech Academy of Sciences Publication Activity Database

    Chodounská, Hana

    2014-01-01

    Roč. 108, S2 (2014), s128 ISSN 0009-2770. [Conference on Isoprenoids /22./. 07.09.2014-10.09.2014, Praha] R&D Projects: GA TA ČR(CZ) TE01020028; GA ČR(CZ) GAP303/12/1464 Institutional support: RVO:61388963 Keywords : hydrogen - deuterium exchange * Wittig reaction Subject RIV: CC - Organic Chemistry

  4. Hydrogen isotope exchange in tungsten irradiated sequentially with low-energy deuterium and protium ions

    NARCIS (Netherlands)

    Alimov, V. K.; Tyburska-Puschel, B.; Hoen, Mhjt; Roth, J.; Hatano, Y.; Isobe, K.; Matsuyama, M.; Yamanishi, T.

    2011-01-01

    Hydrogen isotope exchange in tungsten was investigated at various temperatures both after sequential exposure to low-energy deuterium (D) and protium (H) plasmas and after sequential irradiation with low-energy D and H ions. The methods used were thermal desorption spectroscopy, and the D((3)He,

  5. Hydrogen-Deuterium Exchange of Meteoritic Dicarboxylic Acids During Aqueous Extraction

    Science.gov (United States)

    Fuller, M.; Huang, Y.

    2002-01-01

    This study examines the extent of hydrogen-deuterium exchange on dicarboxylic acids during aqueous extraction. Deuterium enrichment was observed to be a function of diacid structure as well as delta-D. Additional information is contained in the original extended abstract.

  6. Contribution to the study of proteins and peptides structure by hydrogen isotopic exchange

    International Nuclear Information System (INIS)

    Nabedryk-Viala, Eliane.

    1978-01-01

    Development of hydrogen exchange measurement methods to study the structure and the molecular interaction of globular protein molecules in aqueous solution (ribonuclease A, cytochrome c, coupling factors of chloroplasts), in peptide hormones in trifluoroethanol solution (angiotensin II, corticotropin) and in proteins of membranes (rhodopsin) [fr

  7. Anion-exchange synthesis of nanoporous FeP nanosheets as electrocatalysts for hydrogen evolution reaction.

    Science.gov (United States)

    Xu, You; Wu, Rui; Zhang, Jingfang; Shi, Yanmei; Zhang, Bin

    2013-07-28

    Nanoporous FeP nanosheets are successfully synthesized via the anion-exchange reaction of inorganic-organic hybrid Fe18S25-TETAH (TETAH = protonated triethylenetetramine) nanosheets with P ions. The as-prepared nanoporous FeP nanosheets exhibit high electrochemical hydrogen evolution reaction activity in acidic medium.

  8. The folding energy landscape of apoflavodoxin is rugged. hydrogen exchange reveals non-productive misfolded intermediates.

    NARCIS (Netherlands)

    Bollen, Y.J.M.; Kamphuis, M.B.; Mierlo, van C.P.M.

    2006-01-01

    Many native proteins occasionally form partially unfolded forms (PUFs), which can be detected by hydrogen/deuterium exchange and NMR spectroscopy. Knowledge about these metastable states is required to better understand the onset of folding-related diseases. So far, not much is known about where

  9. The folding energy landscape of apoflavodoxin is rugged: Hydrogen exchange reveals nonproductive misfolded intermediates.

    NARCIS (Netherlands)

    Bollen, Y.J.M.; Kamphuis, M.B.; Van Mierlo, C.P.M.

    2006-01-01

    Many native proteins occasionally form partially unfolded forms (PUFs), which can be detected by hydrogen/deuterium exchange and NMR spectroscopy. Knowledge about these metastable states is required to better understand the onset of folding-related diseases. So far, not much is known about where

  10. Analysis and test of a breadboard cryogenic hydrogen/Freon heat exchanger

    Science.gov (United States)

    Desjardins, L. F.; Hooper, J.

    1973-01-01

    System studies required to verify a tube-in-tube cryogenic heat exchanger as optimum for the space shuttle mission are described. Design of the optimum configuration, which could be fabricated from commercially available hardware, is discussed. Finally, testing of the proposed configuration with supercritical hydrogen and Freon 21 is discussed and results are compared with thermal and dynamic analysis.

  11. Exchange of hydrogen isotopes in oxide ceramics at room temperature

    International Nuclear Information System (INIS)

    Suzuki, H.; Morita, K.; Soda, K.

    2001-01-01

    The decay curves of D and up-take curves of H on the exchange of D implanted into Li 2 TiO 3 for H in H 2 O vapor caused by exposure to normal-air at room temperature have been measured as a function of exposure time at different implantation concentrations by means of the elastic recoil detection technique. The re-emission curves of D retained and H up-taken in a specimen by isochronal annealing for 10 min have been also measured. It is found that the thermal re-emission of D and H takes place uniformly over the whole specimen due to local molecular recombination in the bulk and that the re-emission rates of H and D in the near-surface layers are slower than those in the deeper layers. It is also found that the decay of D caused by the D-H exchange takes place rapidly in the beginning and the retained amount of D attains at a constant level and the retained fraction of D are higher as the initial implantation concentrations of D are lower. The decay curves of D and the up-take curves of H have been analysed using the mass balance equations, in which the elementary processes are taken into account according to the exchange model of one way diffusion from the surface into the bulk. It is shown that the solution of the mass balance equations reproduces well the experimental data. The rate constants of the elementary processes determined are discussed. (orig.)

  12. Binary supported nickel catalysts for the deuterium exchange reaction between hydrogen and water vapour

    International Nuclear Information System (INIS)

    Abou El-Nour, F.H.; Belacy, A.

    1982-01-01

    Nickel catalysts supported by Fe 2 O 3 , CuO, MnO and CeO were prepared from the corresponding metal nitrates. Chemical treatment of the catalysts was carried out at room temperature, while thermal treatment was made at 350 0 C. The total surface area of the catalysts was measured by nitrogen adsorption at -195 0 C using the BET equation. The specific metallic surface area was measured by hydrogen chemisorption at liquid nitrogen temperature. The activity of the catalysts was tested for the isotopic exchange reaction of deuterium between hydrogen and water vapour. The results obtained showed that Ni-Fe 2 O 3 , Ni-CuO and Ni-MnO catalysts exhibit catalytic activity for the deuterium exchange between hydrogen and water vapour, while the catalyst supported by CeO has no such activity. (author)

  13. Preparation of Pt-PTFE hydrophobic catalyst for hydrogen-water isotope exchange

    International Nuclear Information System (INIS)

    Li Junhua; Kang Yi; Han Yande; Ruan Hao; Dou Qincheng; Hu Shilin

    2001-01-01

    The hydrophobic catalyst used in the hydrogen-water isotope exchange is prepared with Pt as the active metal, PTFE as the hydrophobic material, active carbon or silicon dioxide as the support. The isotope catalytic exchange reaction between hydrogen and water is carried out in the trickle bed and the effects of different carriers, mass fraction of Pt and PTFE on the catalytic activity are discussed. The experimental results show that the activity of Pt-C-PTFE hydrophobic catalyst with the ratio between PTFE and Pt-C from 1 to 2 is higher than other kinds of catalysts and the overall volume transfer coefficient is increased with the increasing of the hydrogen flow rate and reaction temperature

  14. Hydrogen-water deuterium exchange over metal oxide promoted nickel catalysts

    International Nuclear Information System (INIS)

    Sagert, N.H.; Shaw-Wood, P.E.; Pouteau, R.M.L.

    1975-01-01

    Specific rates have been measured for hydrogen-water deuterium isotope exchange over unsupported nickel promoted with about 20 percent of various metal oxides. The oxides used were Cr 2 O 3 , MoO 2 , MnO, WO 2 -WO 3 , and UO 2 . Nickel surface areas, which are required to measure the specific rates, were determined by hydrogen chemisorption. Specific rates were measured as a function of temperature in the range 353 to 573 K and as a function of the partial pressure of hydrogen and water over a 10-fold range of partial pressure. The molybdenum and tungsten oxides gave the highest specific rates, and manganese and uranium oxides the lowest. Chromium oxide was intermediate, although it gave the highest rate per gram of catalyst. The orders with respect to hydrogen and water over molybdenum oxide and tungsten oxide promoted nickel were consistent with a mechanism in which nickel oxide is formed from the reaction of water with the catalyst, and then is reduced by hydrogen. Over manganese and uranium oxide promoted catalysts, these orders are consistent with a mechanism in which adsorbed water exchanges with chemisorbed hydrogen atoms on the nickel surface. Chromium oxide is intermediate. It was noted that those oxides which favored the nickel oxide route had electronic work functions closest to those of metallic nickel and nickel oxide. (author)

  15. Ab initio molecular dynamics study of the hydrogen-deuterium exchange in bulk lithiumborohydride (LiBH4)

    Science.gov (United States)

    Ramzan, M.; Ahuja, R.

    2009-04-01

    The hydrogen storage is still a challenge for mobile applications. The diffusion of hydrogen in solids is considered as a general model system but there is limited knowledge available for the dynamics of hydrogen in complex hydrides. In this letter, we present a systematic approach to study the hydrogen-deuterium exchange in bulk LiBH4 based on ab initio molecular dynamics. Our results predict the single hydrogen (deuterium) exchange in LiBH4, which supports the experimental results. The self-diffusion constant ⟨D⟩ of deuterium in LiBH4 is around 1.42×10-8 m2 s-1.

  16. Gas phase hydrogen/deuterium exchange of arginine and arginine dipeptides complexed with alkali metals.

    Science.gov (United States)

    Mertens, Laura A; Marzluff, Elaine M

    2011-08-25

    The hydrogen/deuterium (H/D) exchange of protonated and alkali-metal cationized Arg-Gly and Gly-Arg peptides with D(2)O in the gas phase was studied using electrospray ionization quadropole ion trap mass spectrometry. The Arg-Gly and Gly-Arg alkali metal complexes exchange significantly more hydrogens than protonated Arg-Gly and Gly-Arg. We propose a mechanism where the peptide shifts between a zwitterionic salt bridge and nonzwitterionic charge solvated conformations. The increased rate of H/D exchange of the alkali metal complexes is attributed to the peptide metal complexes' small energy difference between the salt-bridge conformation and the nonzwitterionic charge-solvated conformation. Implications for the applicability of this mechanism to other zwitterionic systems are discussed. © 2011 American Chemical Society

  17. Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry.

    Science.gov (United States)

    Wang, Guanbo; Kaltashov, Igor A

    2014-08-05

    Top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection has recently matured to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. However, the scope of the proteins amenable to the analysis by top-down HDX MS still remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. While the limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace, the presence of the disulfide linkages remains a much less forgiving limitation even for the proteins of relatively modest size. To circumvent this problem, we introduce an online chemical reduction step following completion and quenching of the HDX reactions and prior to the top-down MS measurements of deuterium occupancy of individual backbone amides. Application of the new methodology to the top-down HDX MS characterization of a small (99 residue long) disulfide-containing protein β2-microglobulin allowed the backbone amide protection to be probed with nearly a single-residue resolution across the entire sequence. The high-resolution backbone protection pattern deduced from the top-down HDX MS measurements carried out under native conditions is in excellent agreement with the crystal structure of the protein and high-resolution NMR data, suggesting that introduction of the chemical reduction step to the top-down routine does not trigger hydrogen scrambling either during the electrospray ionization process or in the gas phase prior to the protein ion dissociation.

  18. Development of analytical method used for the estimation of potassium amide in liquid ammonia at HWP (Tuticorin)

    International Nuclear Information System (INIS)

    Ramanathan, A.V.

    2007-01-01

    Potassium amide in liquid ammonia is used as a homogeneous catalyst in mono-thermal ammonia-hydrogen isotopic chemical exchange process employed for the manufacture of heavy water. Estimation of concentration of potassium amide in liquid ammonia is vital for checking whether it is sufficient for catalysis in isotopic exchange towers or for purification in purifiers in the Heavy Water Plants. This estimation was carried out earlier by the conventional method involving evaporation of ammonia, decomposition of potassium amide with water and titration of liberated ammonia with sulphuric acid. This method has been replaced by a newly developed method involving direct titration of potassium amide in ammonia with ammonium bromide. This new method is based on the principle that ammonium bromide and potassium amide act as acid and base respectively in the non-aqueous solvent medium, liquid ammonia. This method has not only proved to be an alternative method of estimation of potassium amide in liquid ammonia but also has been serving as a developed analytical method, because it is faster (with fewer steps), more accurate, safer (as it excludes the use of corrosive sulphuric acid needed for the conventional method) and more convenient (as it doesn't need specially designed apparatus and inert gas like dry nitrogen used in the conventional method). (author)

  19. Transitions between elongated conformations of ubiquitin [M+11H]11+ enhance hydrogen/deuterium exchange.

    Science.gov (United States)

    Bohrer, Brian C; Atlasevich, Natalya; Clemmer, David E

    2011-04-21

    Hydrogen/deuterium (H/D) exchange reactions between different elongated conformations of [M + 11H](11+) ions of ubiquitin and D(2)O are studied by a combination of ion mobility spectrometry (IMS) and mass spectrometry techniques. Three conformers (B, C, and D), resolved in the IMS separation, each exchange ∼27 hydrogens upon exposure to 0.06 Torr of D(2)O vapor for ∼35 to 40 ms. However, a region of the IMS spectrum that appears between the C and D states (corresponding to ions that undergo a structural transition during the mobility separation) undergoes substantially more exchanges (∼39 total sites, 44% more than the B, C, and D states). Selection and activation of the individual B, C, and D states reveals that the increased H/D exchange occurs during the transition between structures. Overall, these studies suggest a key process in establishing the maximum exchange levels involves structural transitions, which allow protected sites to be exposed for some fraction of the reaction time. Analysis of changes in exchange levels upon structural transitions can provide insight about common regions of structure that exist in the B, C, and D conformations.

  20. Proton NMR investigation of heme pocket mobility in hemoglobin via hydrogen isotope exchange kinetics

    International Nuclear Information System (INIS)

    Han, K.

    1985-01-01

    Dynamic mobility of heme cavity, the active site of Hb, was investigated by analyzing the hydrogen isotope exchange kinetics of the proximal histidyl ring NH of various kinds of Hbs with the aid of the high field Fourier Transform 1 H NMR spectroscopy. The exchange reaction occurs faster in oxy or R-state Hb than in deoxy or T-state Hb and there exists a good correlation between the oxygen affinity of Hb and the heme pocket mobility reflected in the hydrogen exchange rate. The effect of pH on the exchange is dramatically different for the two subunits of Hb A. Studying the exchange characteristics of mutant Hbs and chemically modified Hbs not only showed the existence of three well-defined localized paths for transmission of conformational changes between different heme pockets through a 1 b 2 subunit interface, but also indicated that the heme pocket mobility is regulated by the quaternary state of Hb as well as by the ligation state of Hb. Finally, the effect of the quaternary state on the heme pocket mobility is separated from that of the ligation by following the exchange reactions in Hbs where only their quaternary structure transition can be achieved without changing their ligation states by adjusting experimental conditions such as adding inositol hexaphosphate

  1. Investigation of the gas-phase hydrogen/deuterium exchange behavior of aromatic dicarboxylic acids in a quadrupole ion trap

    Science.gov (United States)

    Chipuk, Joseph E.; Brodbelt, Jennifer S.

    2007-11-01

    Gas-phase hydrogen/deuterium (H/D) exchange reactions of four deprotonated aromatic dicarboxylic acids (phthalic acid, isophthalic acid, terephthalic acid and 2,6-naphthalic acid) with D2O were performed in a quadrupole ion trap mass spectrometer. Experimental results showed significant differences in the rate and extent of exchange when the relative position of the carboxylic acid groups varied. Spontaneous and near complete exchange of one aromatic hydrogen atom occurred when the carboxylic acid groups were in the meta-position, whereas no additional exchange was observed for either the ortho- or para-isomers or for the structurally similar naphthalic acid. Computational investigations support the participation of several possible exchange mechanisms with the contribution of each relying heavily on the relative orientation of the acid moieties. A relay mechanism that bridges the deprotonation site and the labile hydrogen site appears to be responsible for the H/D exchange of not only the labile hydrogen atom of isophthalic acid, but also for the formation of a stable carbanion and corresponding subsequent exchange of one aromatic hydrogen atom. The impact of hydrogen bonding on the relay mechanism is demonstrated by the reaction of phthalic acid as the extent and rate of reaction are greatly retarded by the favorable interaction of the two carboxylic acid groups. Finally, a flip-flop mechanism is likely responsible for the exchange of both terephthalic acid and 2,6-naphthalic acid where the reactive sites are too remote for exchange via relay.

  2. Asymmetric opening reaction mechanism of Z-DNA base pairs: a hydrogen exchange study

    International Nuclear Information System (INIS)

    Ramstein, J.; Vogt, N.; Leng, M.

    1985-01-01

    With the tritium-Sephadex method, the hydrogen-exchange kinetics of the five NH protons of guanine and cytosine residues in Z-form poly(dG-dC) X poly (dG-dC) were measured as a function of temperature and catalyst concentration. Over the measured temperature range from 0 to 34 degrees C, two classes of protons with constant amplitudes are found. The three protons of the fast class, which were assigned to the guanine amino and imino protons, have an exchange half-time in the minute time range (at 20 degrees C the half-time is 2.5 min) and an activation energy of 18 kcal mol-1. Since these two types of protons exchange at the same rate in spite of their grossly different pK values, the exchange of these protons must be limited by the same nucleic acid conformational change. The two cytosine amino protons of the slow class are especially slow with exchange half-times in the hour time range (at 20 degrees C the exchange half-time is 1 h) and the activation energy is 20 kcal mol-1. The exchange of these two protons is not limited by some nucleic acid conformational change as shown by the marked exchange acceleration of these protons upon addition of 0.2 M imidazole. In addition, we have also reexamined the hydrogen-deuterium exchange kinetics of the amino protons of guanosine cyclic 2',3'-monophosphate by a spectral difference method using a stopped-flow spectrophotometer. The measured kinetic process is monophasic with a rate constant of 3 s-1 at 20 degrees C, which is in the same range as the predicted rate constant of the guanine amino protons

  3. Quantifying hydrogen-deuterium exchange of meteoritic dicarboxylic acids during aqueous extraction

    Science.gov (United States)

    Fuller, M.; Huang, Y.

    2003-03-01

    Hydrogen isotope ratios of organic compounds in carbonaceous chondrites provide critical information about their origins and evolutionary history. However, because many of these compounds are obtained by aqueous extraction, the degree of hydrogen-deuterium (H/D) exchange that occurs during the process needs to be quantitatively evaluated. This study uses compound- specific hydrogen isotopic analysis to quantify the H/D exchange during aqueous extraction. Three common meteoritic dicarboxylic acids (succinic, glutaric, and 2-methyl glutaric acids) were refluxed under conditions simulating the extraction process. Changes in D values of the dicarboxylic acids were measured following the reflux experiments. A pseudo-first order rate law was used to model the H/D exchange rates which were then used to calculate the isotope exchange resulting from aqueous extraction. The degree of H/D exchange varies as a result of differences in molecular structure, the alkalinity of the extraction solution and presence/absence of meteorite powder. However, our model indicates that succinic, glutaric, and 2-methyl glutaric acids with a D of 1800 would experience isotope changes of 38, 10, and 6, respectively during the extraction process. Therefore, the overall change in D values of the dicarboxylic acids during the aqueous extraction process is negligible. We also demonstrate that H/D exchange occurs on the chiral -carbon in 2-methyl glutaric acid. The results suggest that the racemic mixture of 2-methyl glutaric acid in the Tagish Lake meteorite could result from post-synthesis aqueous alteration. The approach employed in this study can also be used to quantify H/D exchange for other important meteoritic compounds such as amino acids.

  4. Polarization measurement of atomic hydrogen beam spin-exchanged with optically oriented sodium atoms

    International Nuclear Information System (INIS)

    Ueno, Akira; Ogura, Kouichi; Wakuta, Yoshihisa; Kumabe, Isao

    1988-01-01

    The spin-exchange reaction between hydrogen atoms and optically oriented sodium atoms was used to produce a polarized atomic hydrogen beam. The electron-spin polarization of the atomic hydrogen beam, which underwent the spin-exchange reaction with the optically oriented sodium atoms, was measured. A beam polarization of -(8.0±0.6)% was obtained when the thickness and polarization of the sodium target were (5.78±0.23)x10 13 atoms/cm 2 and -(39.6±1.6)%, respectively. The value of the spin-exchange cross section in the forward scattering direction, whose scattering angle in the laboratory system was less than 1.0 0 , was obtained from the experimental results as Δσ ex =(3.39±0.34)x10 -15 cm 2 . This value is almost seven times larger than the theoretical value calculated from the Na-H potential. The potential was computed quantum mechanically in the space of the appropriate wave functions of the hydrogen and the sodium atoms. (orig./HSI)

  5. The solubility and isotopic exchange equilibrium for hydrogen isotopes in lithium

    International Nuclear Information System (INIS)

    Smith, F.J.; Land, J.F.; Begun, G.M.; La Gamma de Batistoni, A.M.

    1979-01-01

    The individual solubilities of hydrogen, deuterium and tritium in lithium have been measured as a function of temperature (700 to 1000 0 C) and pressure (0.1 to 760 torr). An expression is derived for the temperature dependence of the individual solubilities when the mole fraction of LiH, LiD or LiT is less than about 0.1. The mutual solubilities and isotopic exchange equilibria of hydrogen and deuterium in lithium have been measured at temperatures between 700 and 900 0 C. Both of the total pressure and individual partial pressures of H 2 , HD, and D 2 have been determined as a function of the mole fraction of LiH and LiD in the lithium solution with varying hydrogen-to-deuterium ratios. In the plateau region, the hydrogen and deuterium could be treated as a single chemical species. However, in the Sieverts region the hydrogen and deuterium dissolved independently of each other. The equilibrium isotope exchange constant varied from 1.28 at 700 0 C to 1.17 to 850 0 C. (U.K.)

  6. On mechanism of low-temperature photoinduced deuterium-hydrogen exchange on silica gel promoted by transition element ions

    International Nuclear Information System (INIS)

    Shuklov, A.D.; Shelimov, B.N.; Kazanskij, V.B.

    1976-01-01

    An investigation has been carried out of the mechanism of the deuterium-hydrogen exchange occurring at 77 0 K on V 5+ /SiO 2 and Co 2+ /SiO 2 which had first been irradiated with UV light in hydrogen. From a study of the isotope exchange kinetics it was established that exchange occurs according to the scheme: Hsub(S) + D 2 reversible Dsub(S) + HD, Dsub(S) + H 2 reversible Hsub(S) + HD, where Hsub(S) and Dsub(S) are hydrogen- and deuterium-containing surface centers. The existence of these centers follows from results on the photosorption of hydrogen and the occurrence of exchange between the photosorbed hydrogen and deuterium from the gas phase

  7. A deuterium-hydrogen exchange catalyst and a method for manufacturing same

    International Nuclear Information System (INIS)

    Adlhart, O.J.; Hindin, S.G.

    1975-01-01

    The invention relates to a catalyst used for inducing the exchange of water hydrogen for deuterium contained in gaseous hydrogen, with a view to enriching water as regards deuterium. That catalyst comprises an inert support, a coating of a hydrophobic resin e.g. polytetrafluoroethylene and a catalytic agent such as platinum on carbon. The catalyst is manufactured by wrapping the inert support by means of a polytetrafluoroethylene emulsion, which is dried. Platinum deposited on carbon is subsequently mixed with another polytetrafluoroethylene emulsion and the mixture obtained is applied on the dried inert support. The invention can be applied to the production of heavy water [fr

  8. The effect of tri-N-acetylglucosamine on hydrogen exchange in hen egg white lysozyme.

    Science.gov (United States)

    Gregory, R B; Dinh, A; Rosenberg, A

    1986-10-25

    Tritium-hydrogen isotope exchange techniques have been employed to study the effect of tri-N-acetylglucosamine binding on the conformational dynamics of hen egg white lysozyme. Numerical Laplace inversion of the data provides exchange rate probability density functions that reveal three overlapping peaks for both the free enzyme and (GlcNAc)3-enzyme complex. Binding of (GlcNAc)3 decreases the exchange rates of all protons to some extent with by far the largest effect being observed for the slow exchanging protons. These have been located, by comparison with neutron diffraction results (Mason, S. A., Bentley, G. A., and McIntyre, G. J. (1984) in Neutrons in Biology (Schoenborn, B. P., ed) pp. 323-334, Plenum Press, New York), within the beta-sheet structure and on helices (8-13), (28-34), and (89-97), that define the edges of the so-called "hydrophobic box" in lysozyme. The regions of the protein that are most affected by binding (GlcNAc)3, as revealed by hydrogen exchange, are found to be quite distinct from the regions observed to undergo conformational changes by x-ray diffraction. Most of these segments of the protein are located at some distance from the (GlcNAc)3-binding site itself. Two segments (the beta-sheet and helix (28-34)) are closely associated with the two active-site carboxylate groups. These results suggest that exchange-stable regions having strong, highly organized hydrogen bonding may have an important role in catalytic function and the differential propagation of conformational and dynamic perturbations caused by ligand binding at distant sites on the protein.

  9. Kinetics of isotopic exchange of [1-3H]saccharides with hydrogen using palladium catalysts

    International Nuclear Information System (INIS)

    Akulov, G.P.; Kayumov, V.G.; Snetkova, E.V.; Kaminskij, Yu.L.

    1988-01-01

    The kinetics was studied of the isotopic exchange of [1- 3 H]saccharides with hydrogen on palladium catalysts. The effect was studied of different factors on the rate of isotopic exchange, e.g., of the composition and structure of saccharides, their concentration in the solution (C), the type of catalyst and of the buffer solution. It was found that by reduced rate of isotopic exchange with hydrogen, all studied saccharides may be arranged into a series independent of the type of catalyst in accordance with the sequence of declining coefficient of relative mobility of l-H atoms during the reaction. Linear dependence was found to exist between the rate constant of the isotopic exchange reaction (r) and the coefficient of relative lability. It was also found that in the range of low concentrations the observed rate constants of isotopic exchange were not dependent on concentration and in the range of higher concentrations, r decreased with increasing C. This character of dependence is justified by the side effect of the processes of sorption on the catalyst. (author). 3 figs., 1 tab., 4 refs

  10. Dual temperature dual pressure water-hydrogen chemical exchange for water detritiation

    Energy Technology Data Exchange (ETDEWEB)

    Sugiyama, Takahiko, E-mail: t-sugiyama@nucl.nagoya-u.ac.jp [Faculty of Engineering, Nagoya University, Fro-cho 1, Chikusa-ku, Nagoya 464-8603 (Japan); Takada, Akito; Morita, Youhei [Faculty of Engineering, Nagoya University, Fro-cho 1, Chikusa-ku, Nagoya 464-8603 (Japan); Kotoh, Kenji [Graduate School of Engineering, Kyushu University, Moto-oka 744, Nishi-ku, Fukuoka 819-0395 (Japan); Munakata, Kenzo [Faculty of Engineering and Resource Science, Akita University, Tegata-gakuen-machi 1-1, Akita 010-8502 (Japan); Taguchi, Akira [Hydrogen Isotope Research Center, University of Toyama, Gofuku 3190, Toyama 930-8555 (Japan); Kawano, Takao; Tanaka, Masahiro; Akata, Naofumi [National Institute for Fusion Science, Oroshi-cho 322-6, Toki, Gifu 509-5292 (Japan)

    2015-10-15

    Experimental and analytical studies on hydrogen-tritium isotope separation by a dual temperature dual pressure catalytic exchange (DTDP-CE) with liquid phase chemical exchange columns were carried out in order to apply it to a part of the water detritiation system for DEMO fuel cycle. A prototype DTDP-CE apparatus was successfully operated and it was confirmed that tritium was separated by the apparatus as significantly distinguishable. A calculation code was developed based on the channeling stage model. The values of separation factors and the effects of some operating parameters were well predicted by the separative analyses with the code.

  11. Solvation effects in kinetics of reactions of deuterium-hydrogen exchange with carboxylic acid

    International Nuclear Information System (INIS)

    Serebryanskaya, A.I.; Kurenkova, V.N.; Shatenshtejn, A.I.

    1980-01-01

    A comparative study of the kinetic of the reaction of deuterium-hydrogen exchange of 1,3-dideuteroazulene and 2,4,6-trideuterotrimethoxybenzene with acetic acid is carried out at a wide variation of its concentration. Peculiarities of the competitive effect of electron-donor and polar properties of aprotic solvents are pointed out. Comparison with analogous data obtained earlier for deuterium exchange of 1,4-D 2 -durene with trifluoroacetic acid permitted to find role of effects of specific and nonspecific solvation depending on medium properties and also on dissociation constant and polarity of carboxylic acid

  12. Hydrogen/deuterium exchange mass spectrometry applied to IL-23 interaction characteristics: potential impact for therapeutics.

    Science.gov (United States)

    Iacob, Roxana E; Krystek, Stanley R; Huang, Richard Y-C; Wei, Hui; Tao, Li; Lin, Zheng; Morin, Paul E; Doyle, Michael L; Tymiak, Adrienne A; Engen, John R; Chen, Guodong

    2015-04-01

    IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.

  13. Gaseous exchange reaction of deuterium between hydrogen and water on hydrophobic catalyst supporting platinum

    International Nuclear Information System (INIS)

    Izawa, Hirozumi; Isomura, Shohei; Nakane, Ryohei.

    1979-01-01

    The deuterium exchange reaction between hydrogen and water in the gas phase where the fed hydrogen gas is saturated with water vapor is studied experimentally by use of the proper hydrophobic catalysts supporting platinum. It is found that the activities of those catalysts for this reaction system are very high compared with the other known ones for the systems in which gas and liquid should coexist on catalyst surfaces, and that the apparent catalytic activity becomes larger as the amount of platinum supported on a catalyst particle increases. By analyses of the data the following informations are obtained. The exchange reaction can be expressed by a first order reversible reaction kinetics. The pore diffusion in the catalyst particles has significant effect on the overall reaction mechanisms. (author)

  14. Experimental determination of reaction rates of water. Hydrogen exchange of tritium with hydrophobic catalysts

    International Nuclear Information System (INIS)

    Bixel, J.C.; Hartzell, B.W.; Park, W.K.

    1976-01-01

    This study was undertaken to obtain data needed for further development of a process for the enrichment and removal of tritium from the water associated with light-water reactors, fuel-reprocessing plants, and tritium-handling laboratories. The approach is based on the use of antiwetting, hydrophobic catalysts which permit the chemical exchange reactions between liquid water and gaseous hydrogen in direct contact, thus eliminating problems of catalyst deactivation and the complexity of reactor design normally associated with current catalytic-detritiation techniques involving gas-phase catalysis. An apparatus and procedure were developed for measuring reaction rates of water-hydrogen chemical exchange with hydrophobic catalysts. Preliminary economic evaluations of the process were made as it might apply to the AGNS fuel reprocessing plant

  15. Comparison of Methods for Computing the Exchange Energy of quantum helium and hydrogen

    International Nuclear Information System (INIS)

    Cayao, J. L. C. D.

    2009-01-01

    I investigate approach methods to find the exchange energy for quantum helium and hydrogen. I focus on Heitler-London, Hund-Mullikan, Molecular Orbital and variational approach methods. I use Fock-Darwin states centered at the potential minima as the single electron wavefunctions. Using these we build Slater determinants as the basis for the two electron problem. I do a comparison of methods for two electron double dot (quantum hydrogen) and for two electron single dot (quantum helium) in zero and finite magnetic field. I show that the variational, Hund-Mullikan and Heitler-London methods are in agreement with the exact solutions. Also I show that the exchange energy calculation by Heitler-London (HL) method is an excellent approximation for large inter dot distances and for single dot in magnetic field is an excellent approximation the Variational method. (author)

  16. HOGEN{trademark} proton exchange membrane hydrogen generators: Commercialization of PEM electrolyzers

    Energy Technology Data Exchange (ETDEWEB)

    Smith, W.F.; Molter, T.M. [Proton Energy Systems, Inc., Rocky Hill, CT (United States)

    1997-12-31

    PROTON Energy Systems` new HOGEN series hydrogen generators are Proton Exchange Membrane (PEM) based water electrolyzers designed to generate 300 to 1000 Standard Cubic Feet Per Hour (SCFH) of high purity hydrogen at pressures up to 400 psi without the use of mechanical compressors. This paper will describe technology evolution leading to the HOGEN, identify system design performance parameters and describe the physical packaging and interfaces of HOGEN systems. PEM electrolyzers have served US and UK Navy and NASA needs for many years in a variety of diverse programs including oxygen generators for life support applications. In the late 1970`s these systems were advocated for bulk hydrogen generation through a series of DOE sponsored program activities. During the military buildup of the 1980`s commercial deployment of PEM hydrogen generators was de-emphasized as priority was given to new Navy and NASA PEM electrolysis systems. PROTON Energy Systems was founded in 1996 with the primary corporate mission of commercializing PEM hydrogen generators. These systems are specifically designed and priced to meet the needs of commercial markets and produced through manufacturing processes tailored to these applications. The HOGEN series generators are the first step along the path to full commercial deployment of PEM electrolyzer products for both industrial and consumer uses. The 300/1000 series are sized to meet the needs of the industrial gases market today and provide a design base that can transition to serve the needs of a decentralized hydrogen infrastructure tomorrow.

  17. Catalytic heat exchangers for small-scale production of hydrogen - feasibility study

    Energy Technology Data Exchange (ETDEWEB)

    Silversand, F. [Catator AB, Lund (Sweden)

    2002-02-01

    A feasibility study concerning heat-exchanger reactors in small-scale production of hydrogen has been performed on the request of Svenskt Gastekniskt Center AB and SWEP International AB. The basic idea is to implement different catalysts into brazed plate-type heat exchangers. This can be achieved by installing catalytic cylinders in the inlet-and outlet ports of the heat exchangers or through treatment of the plates to render them catalytically active. It is also possible to sandwich catalytically active wire meshes between the plates. Experiments concerning steam reforming of methanol and methane have been performed in a micro-reactor to gather kinetic data for modelling purposes. Performance calculations concerning heat exchanger reactors have then been conducted with Catator's generic simulation code for catalytic reactors (CatalystExplorer). The simulations clearly demonstrate the technical performance of these reactors. Indeed, the production rate of hydrogen is expected to be about 10 nm{sup 3}/h per litre of heat exchanger. The corresponding value for a conventional steam-reforming unit is about 1 nm{sup 3}/h or less per litre of reactor volume. Also, the compactness and the high degree of integration together with the possibilities of mass production will give an attractive cost for such units. Depending on the demands concerning the purity of the hydrogen it is possible to add secondary catalytic steps like water-gas shifters, methanation and selective oxidation, into a one-train unit, i.e. to design an all-inclusive design. Such reactors can be used for the supply of hydrogen to fuel cells. The production cost for hydrogen can be cut by 60 - 70% through the utilisation of heat exchanger reactors instead of conventional electrolysis. This result is primarily a result of the high price for electricity compared to the feed stock prices in steam reforming. It is important to verify the performance calculations and the simulation results through

  18. Probing conformational changes in rhodopsin using hydrogen-deuterium exchange coupled to mass spectrometry.

    Science.gov (United States)

    Orban, Tivadar; Tsybovsky, Yaroslav

    2015-01-01

    Hydrogen-deuterium exchange coupled to mass spectrometry is a powerful tool to evaluate changes in protein conformation between two or more states. Here, we describe a complete methodology that can be used to assess conformational changes in rhodopsin accompanying its transition from the inactive to activated state upon light exposure. This approach may be employed to investigate the structure and conformational changes of various membrane proteins.

  19. Hydrogen-deuterium exchange of weak carbon acids under phase-transfer catalysis conditions

    International Nuclear Information System (INIS)

    Feldman, D.; Halpern, M.; Rabinovitz, M.

    1985-01-01

    A practical method for hydrogen-deuterium exchange is obtained via extractive hydroxide ion initiated phase-transfer catalysis. The reaction of NaOD/D 2 O system allows the easy production of compounds that otherwise would require very strong bases and aprotic solvents. The strong basicity of OD - anion is attributable to its relative freedom from water molecules when OD - is extracted into the depth of the organic layer. 22 references, 1 table

  20. Shape of the Hα emission line in non resonant charge exchange in hydrogen plasmas

    International Nuclear Information System (INIS)

    Susino Bueno, A.; Zurro Hernandez, B.

    1977-01-01

    The Hα line shape emitted from a maxwellian hydrogen plasma and produced by non resonant change exchange has been calculated. Its explicit shape depends on the ion temperature, on background neutral energy and on the relative shape of the collision cross section. A comparison between theoretical and experimental shapes of the Hα line is carried out to check the model and to deduce the ion plasma temperature. (author) [es

  1. On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS)*

    OpenAIRE

    Quanico, Jusal; Franck, Julien; Salzet, Michel; Fournier, Isabelle

    2016-01-01

    Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and...

  2. Hydrogen isotope exchange and conditioning in graphite limiters used in TFTR

    International Nuclear Information System (INIS)

    LaMarche, P.H.; Dylla, H.F.; McCarthy, P.J.; Ulrickson, M.

    1986-02-01

    Isotopic exchange experiments performed in TFTR are used to examine the outgassing and diffusive properties of graphite used as the plasma limiter. Changeover from hydrogen to deuterium for different periods ranges from approx.600 to 60 plasma discharges, which appears to be correlated to the limiter temperature. We present a simple analytical model that predicts a fast transient (approx.10 plasma discharges) changeover where the deuterium fueling dilutes the adsorbed and near-surface hydrogen, and a slowly changing term where bulk hydrogen diffuses to the surface. Using this model we can extract an activation energy for diffusion of 0.15 +- 0.02 eV. We hypothesize that interpore diffusion for this porous (approx.15%) material is consistent with our observations. 19 refs

  3. Hydrogen isotope exchange and conditioning in graphite limiters used in the Tokamak Fusion Test Reactor

    International Nuclear Information System (INIS)

    LaMarche, P.H.; Dylla, H.F.; McCarthy, P.J.; Ulrickson, M.

    1986-01-01

    Isotopic exchange experiments performed in the Tokamak Fusion Test Reactor (TFTR) are used to examine the outgassing and diffusive properties of graphite used as the plasma limiter. Changeover from hydrogen to deuterium for different periods ranges from approx.600 to 60 plasma discharges, which appears to be correlated in the limiter temperature. We present a simple analytical model that predicts a fast transient (approx.10 plasma discharges) changeover, where the deuterium fueling dilutes the adsorbed and near surface hydrogen, and a slowly changing term where bulk hydrogen diffuses to the surface. Using this model we can extract an activation energy for diffusion of 0.15 +- 0.02 eV. We hypothesize that interpore diffusion for this porous (approx.15%) material is consistent with our observations

  4. Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development – A review

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Bin, E-mail: dengbin@yorku.ca [Chemistry Department, York University, 4700 Keele Street, Toronto, ON, M3J 1P3 (Canada); The Centre for Research in Mass Spectrometry, York University, Toronto, ON, M3J1P3 (Canada); Lento, Cristina, E-mail: clento@yorku.ca [Chemistry Department, York University, 4700 Keele Street, Toronto, ON, M3J 1P3 (Canada); The Centre for Research in Mass Spectrometry, York University, Toronto, ON, M3J1P3 (Canada); Wilson, Derek J., E-mail: dkwilson@yorku.ca [Chemistry Department, York University, 4700 Keele Street, Toronto, ON, M3J 1P3 (Canada); The Centre for Research in Mass Spectrometry, York University, Toronto, ON, M3J1P3 (Canada)

    2016-10-12

    Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways. In manufacturing and development, HDX-MS can validate storage formulations and manufacturing processes for various biotherapeutics. Currently, HDX-MS is being refined to provide additional coverage, sensitivity and structural specificity and implemented on the millisecond timescale to reveal residual structure and dynamics in disordered domains and intrinsically disordered proteins. - Highlights: • The pharmaceuticals industry is increasingly shifting to protein therapeutics. • Hydrogen deuterium exchange mass spectrometry is uniquely well suited to support biopharmaceutical development. • Applications for hydrogen deuterium exchange span drug discovery, development and manufacturing. • Future developments will allow improved sensitivity, structural resolution and a broader range of dynamics to be monitored.

  5. Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

    Science.gov (United States)

    Burkitt, William; Domann, Paula; O'Connor, Gavin

    2010-01-01

    Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247–253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the CH2 and CH3 domains. PMID:20162626

  6. Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development – A review

    International Nuclear Information System (INIS)

    Deng, Bin; Lento, Cristina; Wilson, Derek J.

    2016-01-01

    Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways. In manufacturing and development, HDX-MS can validate storage formulations and manufacturing processes for various biotherapeutics. Currently, HDX-MS is being refined to provide additional coverage, sensitivity and structural specificity and implemented on the millisecond timescale to reveal residual structure and dynamics in disordered domains and intrinsically disordered proteins. - Highlights: • The pharmaceuticals industry is increasingly shifting to protein therapeutics. • Hydrogen deuterium exchange mass spectrometry is uniquely well suited to support biopharmaceutical development. • Applications for hydrogen deuterium exchange span drug discovery, development and manufacturing. • Future developments will allow improved sensitivity, structural resolution and a broader range of dynamics to be monitored.

  7. Detection of carbanions in aqueous solution by nuclear magnetic resonance spectroscopy. Hydrogen--deuterium exchange reactions of aromatic mercaptals

    International Nuclear Information System (INIS)

    Pesek, J.J.; Mitchell, S.J.

    1976-01-01

    Carbanions are detected in basic aqueous solution by hydrogen-deuterium exchange at the acidic carbon. Both proton and carbon-13 nuclear magnetic resonance spectroscopy can be used to observe the H-D exchange. The proton nmr spectra can be used to quantitatively determine the exchange rate. Mercaptals are studied because the two sulfur atoms adjacent to the acidic carbon favor carbanion formation. It is found that only when the acidic carbon has an aromatic substituent can hydrogen-deuterium exchange be observed

  8. Production of highly spin-polarized atomic hydrogen and deuterium by spin-exchange

    International Nuclear Information System (INIS)

    Redsun, S.G.

    1990-01-01

    The first part of this work is a study of the production of highly spin-polarized atomic hydrogen and deuterium by spin-exchange optical pumping. A tunable ring dye laser is used to polarize rubidium atoms by optical pumping. The cell containing the rubidium vapor is coated with paraffin in order to reduce spin relaxation due to wall collisions. Hydrogen gas is dissociated in an inductive discharge and flows continuously through the cell, in which the hydrogen atoms are polarized by spin-exchange collisions with the polarized rubidium atoms. The hydrogen polarization is determined by a combination of fluorescence monitoring and magnetic resonance spectroscopy. Atomic hydrogen polarization as high as 2 z > H = 0.72(6) has been observed, which is the highest degree of polarization yet produced by this method. However, the polarization may be limited to this value due to the depolarization of the rubidium by radiation trapping. The spin-relaxation rate of atomic hydrogen on a paraffin-coated cell is also measured for the first time, and corresponds to about 3,800 wall bounces before electron-spin randomization. The second part of this work is a theoretical analysis of the problem of radiation trapping in a dense optically pumped alkali vapor. A Monte Carlo routine is used to simulate the trajectories of multiply scattered photons. The average spin angular momentum transfer from the photons to the vapor is used to determine the equilibrium polarization of the vapor as a function of the alkali density and the frequency of the pumping light

  9. Hydrogen isotope exchange of organic compounds in dilute acid at elevated temperatures

    International Nuclear Information System (INIS)

    Werstiuk, N.H.

    1987-01-01

    Introduction of one or more deuterium (or tritium) atoms into organic molecules can be accomplished in many ways depending on the nature of the substrate and the extent and sterochemistry of deuteriation or tritiation required. Some of the common methods include acid- and base-catalyzed exchange of carbonyl compounds, metal hydride reductions, dissolving metal reductions, catalytic reduction of double bonds, chromatographic exchange, homogeneous and heterogeneous metal-catalyzed exchange, base-catalyzed exchange of carbon acids other than carbonyl compounds and acid-catalyzed exchange via electrophilic substitution. Only the latter three methods have been used for perdeuteriation of organic compounds. A very useful compendium of labeling methods with examples has been available to chemists for some time. Although metal-catalyzed exchange has been used extensively, the method suffers from some deficiencies: irreproducibility of catalyst surfaces, catalyst poisoning, side reactions such as coupling and hydrogenolysis of labile groups and low deuterium incorporation. Usually a number of cycles are required with fresh catalyst and fresh deuterium source to achieve substantial isotope incorporation. Acid-catalyzed exchange of aromatics and alkenes, strongly acidic media such as liquid DBr, concentrated DBr, acetic acid/stannic chloride, concentrated D 3 PO 4 , concentrated DC1, D 3 PO 4 /BF 3 SO 2 , 50-80% D 2 SO 4 and DFSO 4 /SbF 5 at moderate temperatures (<100 degrees) have been used to effect exchange. The methods are not particularly suitable for large scale deuteriations because of the cost and the fact that the recovery and upgrading of the diluted deuterium pool is difficult. This paper describes the hydrogen isotope exchange of a variety of organic compounds in dilute aqueous acid (0.1-0.5 M) at elevated temperatures (150-300 degrees)

  10. Dynamic diversity of synthetic supramolecular polymers in water as revealed by hydrogen/deuterium exchange

    Science.gov (United States)

    Lou, Xianwen; Lafleur, René P. M.; Leenders, Christianus M. A.; Schoenmakers, Sandra M. C.; Matsumoto, Nicholas M.; Baker, Matthew B.; van Dongen, Joost L. J.; Palmans, Anja R. A.; Meijer, E. W.

    2017-05-01

    Numerous self-assembling molecules have been synthesized aiming at mimicking both the structural and dynamic properties found in living systems. Here we show the application of hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to unravel the nanoscale organization and the structural dynamics of synthetic supramolecular polymers in water. We select benzene-1,3,5-tricarboxamide (BTA) derivatives that self-assemble in H2O to illustrate the strength of this technique for supramolecular polymers. The BTA structure has six exchangeable hydrogen atoms and we follow their exchange as a function of time after diluting the H2O solution with a 100-fold excess of D2O. The kinetic H/D exchange profiles reveal that these supramolecular polymers in water are dynamically diverse; a notion that has previously not been observed using other techniques. In addition, we report that small changes in the molecular structure can be used to control the dynamics of synthetic supramolecular polymers in water.

  11. Hydrogen water deuterium exchange studies on palladium on activated charcoal hydrophobic catalyst (Preprint No. CA-20)

    International Nuclear Information System (INIS)

    Dubey, Krishna; Malaikar, N.L.; Pushpangathan, C.K.

    1989-04-01

    Deuterium exchange between hydrogen gas and water is one of the most promising processes for heavy water production. In production of heavy water, separation factor and energy cost are two main parameters which govern the economic effectiveness of a process. Out of the chemical exchange process, H 2 -H 2 O exchange has higher separation factor at a given temperature. Even though the separation factor is high, major disadvantage in the process is that a catalyst is required. Group VIII metals are most suitable catalysts for hydrogenation, dehydrogenation and hydrogenolysis because of their ability to chemisorb H 2 dissociatively. Even among VIII2 triad, Pt has the highest activity. A highly active Pt catalyst has a reported half life of 4 seconds. As Pd is cheaper than Pt, studies have been carried out using active Pd as catalyst for H 2 -H 2 O exchange. It is observed that: (1)at metal concentration of 0.3%, Pd shows the optimum catalytic activity, (2)a highly active Pd is found to have a half life of 5 minutes, and (3)addition of α-alumina enhances the catalytic activity. (author). 6 refs., 5 figs

  12. Model for the simulation of catalytic isotope exchange between tritiated water and hydrogen/deuterium gas

    International Nuclear Information System (INIS)

    Cristescu, Ioana R.; Cristescu, I.; Bornea, Anisia; Penzhorn, R.-D.; Tamm, U.

    2001-01-01

    The objectives of the models presented in this paper are to simulate the deuterium enrichment performances of several catalytic exchange columns under test at the Tritium Laboratory in Karlsruhe (TLK). The models and the corresponding software are presently being verified by experiments aimed to provide the data required for the design of large isotopic exchange columns such as needed for the recovery of tritium from waste water generated during the operation of the tritium facilities of fusion machines. In the ongoing TLK experiments the transfer of deuterium takes place in a counter current isotopic exchange column. Pre-heated water is fed into the column from the top and deuterated hydrogen from the bottom. Condensed water vapor and deuterium-depleted hydrogen are removed from the top and deuterium enriched water from the bottom and analyzed by standard procedures. To describe the performance of the separation process two approaches were considered: a graphical approach (classical) that uses the operating line and equilibrium curve for the process and an analytical approach that solves the transport equations characterizing the isotopic exchange process. (authors)

  13. Isotope separation factor and isotopic exchange rate between hydrogen and deuterium of palladium

    International Nuclear Information System (INIS)

    Fukada, S.; Fuchinoue, K.; Nishikawa, M.

    1995-01-01

    An isotopic exchange experiment was performed using a Pd particle bed for a fundamental study of hydrogen isotope separation. The isotope separation factor and the rate constant of the isotopic exchange reaction between gaseous deuterium and hydride were determined from fitting numerical calculations to experimental effluent curves of the Pd bed. The separation factors under the condition of a dilute deuterium concentration were correlated with the relation of α H-D = exp(-0.121 + 228/T) and were independent of the total hydrogen pressure. The rate-determining step of the overall isotopic exchange reaction at T > 300 K was diffusion in the pore of Pd particles. The step at T < 300 K was estimated to be diffusion in the β-phase Pd, although there is a little possibility of isotopic exchange reaction based on Bonhoeffer-Farcus mechanism. The height equivalent to a theoretical plate, HETP, was correlated as a function of the interstitial fluid velocity in the bed. (orig.)

  14. Hydrogen/deuterium exchange of multiply-protonated cytochrome c ions

    International Nuclear Information System (INIS)

    Wood, T.D.; Guan, Ziqiang; O'Connor, P.B.

    1995-01-01

    Low resolution measurements show gaseous multiply-protonated cytochrome c ions undergo hydrogen/deuterium (H/D) exchange with pseudo first-order kinetics at three distinct exchange levels, suggesting the co-existence of gaseous protein conformations. Although exchange levels first increase with increasing charge values, they decrease at the highest charge values, consistent with solution-phase behavior of cytochrome c, where the native structure unfolds with decreasing pH until folding into a compact A-state at lowest pH. High resolution measurements indicate the presence of at least six H/D exchange levels. Infrared (IR) laser heating and fast collisions via quadrupolar excitation (QE) increase H/D exchange levels (unfolding) while charge-stripping ions to lower charge values can increase or decrease H/D exchange levels (unfolding or folding). Wolynes has suggested studying proteins in vacuo could play an important role in delineating the contributions various forces play in the protein folding process, provided appropriate comparisons can be made between gas-phase and solution-phase structures

  15. In-ESI source hydrogen/deuterium exchange of carbohydrate ions.

    Science.gov (United States)

    Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Nikolaev, Eugene

    2014-03-04

    We present the investigation of hydrogen/deuterium (H/D) exchange of carbohydrates ions occurring in the electrospray ion source. The shape of the deuterium distribution was observed to be considerably dependent on the temperature of the ion transfer tube and the solvent used. If deuterated alcohol (EtOD or MeOD) or D2O/deuterated alcohol is used as an electrospray solvent, then for high temperatures (>350 °C), intensive back exchange is observed, resulting in ∼30% depth of the deuterium exchange. At low temperatures (exchange is weaker and the depth of the deuterium exchange is ∼70%. In the intermediate temperature region (∼250 °C), the deuterium distribution is unusually wide for methanol and bimodal for ethanol. The addition of 1% formic acid results in low (∼30%) depth of the deuterium exchange for any temperature in the operating region. The bimodal distribution for the ethanol can be possibly explained by the presence of differently folded gas-phase ions of carbohydrates.

  16. Ion mobility spectrometry-hydrogen deuterium exchange mass spectrometry of anions: part 1. Peptides to proteins.

    Science.gov (United States)

    Donohoe, Gregory C; Khakinejad, Mahdiar; Valentine, Stephen J

    2015-04-01

    Ion mobility spectrometry (IMS) coupled with hydrogen deuterium exchange (HDX)-mass spectrometry (MS) has been used to study the conformations of negatively-charged peptide and protein ions. Results are presented for ion conformers of angiotensin 1, a synthetic peptide (SP), bovine insulin, ubiquitin, and equine cytochrome c. In general, the SP ion conformers demonstrate a greater level of HDX efficiency as a greater proportion of the sites undergo HDX. Additionally, these ions exhibit the fastest rates of exchange. Comparatively, the angiotensin 1 ions exhibit a lower rate of exchange and HDX level presumably because of decreased accessibility of exchange sites by charge sites. The latter are likely confined to the peptide termini. Insulin ions show dramatically reduced HDX levels and exchange rates, which can be attributed to decreased conformational flexibility resulting from the disulfide bonds. For the larger ubiquitin and protein ions, increased HDX is observed for larger ions of higher charge state. For ubiquitin, a conformational transition from compact to more elongated species (from lower to higher charge states) is reflected by an increase in HDX levels. These results can be explained by a combination of interior site protection by compact conformers as well as decreased access by charge sites. The elongated cytochrome c ions provide the largest HDX levels where higher values correlate with charge state. These results are consistent with increased exchange site accessibility by additional charge sites. The data from these enhanced IMS-HDX experiments are described in terms of charge site location, conformer rigidity, and interior site protection.

  17. General Tritium Labelling of Gentamicin C by catalytic hydrogen exchange Reaction with Tritiated Water

    International Nuclear Information System (INIS)

    Suarez, C.; Diaz, D.; Paz, D.

    1991-01-01

    Gentamicin C was labelled with tritium by means of a PtO2 catalyzed hydrogen exchange reaction. Under the conditions of the exchange (100 mg of gentamicin, basic form, 0,3 ml H2O-3H, and 50 mg of prereduced PtO2) the radiochemical yield was 0,24, 0,38 and 0,48 % at 120 degree celsius, for 8, 16 and 24 hours respectively. Chemical yield for purified gentamicin was about 60 %. Purification was accomplished with a cellulose column eluted with the lower phase of chloroform-methanol 17 % ammonium hydroxide (2:1:1, v/v) . Chemical purity, determined by HPLC, was 96,5 % and radiochemical one was 95. Main exchange degradation products show biological activity. (Author) 12 refs

  18. Hydrogen-deuterium (h-d) exchange reaction of warfarin in D(2)O solution.

    Science.gov (United States)

    Tode, Chisato; Takeuchi, Atsuko; Iwakawa, Seigo; Tatsumi, Akitoshi; Sugiura, Makiko

    2009-07-01

    To prove the presence of a hydrogen-deuterium (H-D) exchange reaction, (1)H- and (13)C-NMR spectra of warfarin were measured in solvents containing D(2)O and H(2)O. In D(2)O or D(2)O/dimethyl sulfoxide (DMSO)-d(6) solvent, signal pattern changes were observed on H12 and H11 as well as 14 methyl protons over time while no changes were observed on H(2)O or H(2)O/DMSO-d(6) solvent. The observed changes in the solvents containing D(2)O were concluded to be caused by the H-D exchange reaction on H12, the process of CH(2)-->CHD-->CD(2). MS spectroscopy also confirmed these H-D exchanges. The kinetics of this reaction were analyzed as the successive reaction, and the mechanism was also proposed.

  19. Zinc oxide and chromia as catalysts for the isomerization of butene, the hydrogenation of ethylene, and the isotopic exchange and allotropic conversion of hydrogen

    International Nuclear Information System (INIS)

    Conner, W.C. Jr.

    1973-01-01

    Hydrogenation of olefins has been studied over metals and metal oxides. Over metals the following observations generalize the characteristics of hydrogenation and isomerization. Metal hydrogenation catalysts are effective for H 2 -D 2 exchange (and para hydrogen conversion) under the same conditions as they effect olefin hydrogenation. This suggests that hydrogen ''activation'' involves formation of hydrogen atoms as a surface intermediate. Addition of deuterium to light ethylene leads to ethane products of the form C 2 H/sub 6-x/D/sub x/ (where 0 less than or equal to x less than or equal to 6). This is a result of the reversal of the alkyl (C 2 H 5 *) formation on the surface. Moreover, efficient isomerization of olefins require hydrogen as a co-catalyst. Both these observations suggest that alkyl formation and its reversal play a major role in hydrogenation and related reactions over metals. In this work it is found that zinc oxide catalyzes the deuteration of ethylene to dideuterioethane selectivity. Furthermore, the hydrogenation of ethylene using mixtures of hydrogen and deuterium indicate that hydrogenation occurs in such a manner as to reflect the molecular identity of the gas phase in the product ethane

  20. Factors determining the activity of catalysts of different chemical types in the oxidation of hydrogen. III. Oxidation and isotopic exchange of hydrogen on tungsten carbide

    International Nuclear Information System (INIS)

    Il'chenko, N.I.; Dolgikh, L.Y.; Golodets, G.I.

    1985-01-01

    It is explained how the oxidation of hydrogen on tungsten carbide at 653 K comes about via two basic mechanisms. In an excess of H 2 , the predominant mechanism involves the reaction of O 2 with dissociated, adsorbed hydrogen while at low concentrations of hydrogen it is the reaction of H 2 with adsorbed oxygen that makes the basic contribution to the observed rate. A model is proposed which also describes satisfactorily the rate of the simultaneously occurring isotopic exchange of hydrogen with deuterium

  1. Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry

    OpenAIRE

    Masson, Glenn R.; Maslen, Sarah L.; Williams, Roger L.

    2017-01-01

    Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen in...

  2. Exchange of deuterium for hydrogen during the reaction of dimethylamine-N-d-borane with halogenating agents

    International Nuclear Information System (INIS)

    Myers, W.H.; Ryschkewitsch, G.E.

    1978-01-01

    During the reaction of dimethylamine-N-d-borane with chlorine considerable exchange of hydrogen for deuterium on nitrogen occurred. An extensive investigation of the halogenation reactions of dimethylamine-N-d-borane indicated that the exchange process occurred via the loss of deuterium chloride from a molecule activated as a result of halogenation. The extent of substitution of hydrogen for deuterium in the products of these reactions was estimated by comparing NMR and IR spectra

  3. Study of Hydrogen Consumption by Control System in Proton Exchange Membrane Fuel Cell

    International Nuclear Information System (INIS)

    Ros Emilia Rosli; Edy Herianto Majlan; Siti Afiqah Abd Hamid; Wan Ramli Wan Daud; Ramizi Mohamed; Dedi Rohendi

    2016-01-01

    Efficient operation results from a proper control strategy. In the operation and performance of a Proton Exchange Membrane Fuel Cell (PEMFC), the hydrogen gas flow rate is one of the most essential control parameter in addition to operating pressure, water management, temperature and humidity. This is because of the high cost and amount of energy are required to produce the purity hydrogen gas. In this paper, a Proportional Integral Derivative (PID) feedback control system is used to control the hydrogen flow rate. A strategy is adapted to balance the hydrogen use based on the loading requirements, especially during start-ups and sudden power demands. This system is implemented using National Instrument (NI) devices powered by the LabVIEW program. This is due to its simplicity and customization flexibility for measuring, processing and recording data. Designed structure allows the real-time implementation of a robust control law that is able to address the related nonlinearities and uncertainties without incurring a heavy computational load for the controller algorithm. While it facilitating a fast sampling rate according to the needs of the power system. Test results from the controller show that the new fuel control system provides good performance by reducing the amount of wasted hydrogen gas compared with that of the previous open loop system by 30 % to over 80 % saved by the varied load. This improvement is beneficial for any PEMFC that experiences fluctuating power demand, especially for vehicle applications. (author)

  4. Measurement of a hyperfine-induced spin-exchange frequency shift in atomic hydrogen

    International Nuclear Information System (INIS)

    Walsworth, R.L.; Silvera, I.F.; Mattison, E.M.; Vessot, R.F.C.

    1992-01-01

    We have measured a hyperfine-induced spin-exchange frequency shift in the atomic-hydrogen ground-state hyperfine transition. A recent quantum-mechanical treatment of low-energy hydrogen-hydrogen scattering by Koelman et al. [Phys. Rev. A 38, 3535 (1988)] predicts such frequency shifts to become large at low temperature, and to affect the performance of atomic clocks such as the cryogenic hydrogen maser. The experiment reported here was performed with a hydrogen maser operating near room temperature, where the reported hyperfine effects are predicted to be small, but measurable. Using an adiabatic fast passage (AFP) technique to vary the incoming atomic population in the masing states from approximately 100% (AFP on) to 50% (AFP off), we determined the change in the dimensionless hyperfine-induced frequency-shift parameter Ω to be Ω on -Ω off =5.38 (1.06)x10 -4 . The theoretical prediction at this temperature is Ω on -Ω off =-0.76x10 -4 to -1.12x10 -4 , for the range of masing-state populations used in the present experiment. We review the relevant theory, report our experimental method and results, and discuss possible reasons for the discrepancy between experiment and theory

  5. Conformation-Specific IR and UV Spectroscopy of the Amino Acid Glutamine: Amide-Stacking and Hydrogen Bonding in AN Important Residue in Neurodegenerative Diseases

    Science.gov (United States)

    Walsh, Patrick S.; Dean, Jacob C.; Zwier, Timothy S.

    2014-06-01

    Glutamine plays an important role in several neurodegenerative diseases including Huntington's disease (HD) and Alzheimer's disease (AD). An intriguing aspect of the structure of glutamine is its incorporation of an amide group in its side chain, thereby opening up the possibility of forming amide-amide H-bonds between the peptide backbone and side chain. In this study the conformational preferences of two capped gluatamines Z(carboxybenzyl)-Glutamine-X (X=OH, NHMe) are studied under jet-cooled conditions in the gas phase in order to unlock the intrinsic structural motifs that are favored by this flexible sidechain. Conformational assignments are made by comparing the hydride stretch ( 3100-3700 cm-1) and amide I and II ( 1400-1800 cm-1) resonant ion-dip infrared spectra with predictions from harmonic frequency calculations. Assigned structures will be compared to previously published results on both natural and unnatural residues. Particular emphasis will be placed on the comparison between glutamine and unconstrained γ-peptides due to the similar three-carbon spacing between backbone and side chain in glutamine to the backbone spacing in γ-peptides. The ability of the glutamine side-chain to form amide stacked conformations will be a main focus, along with the prevalence of extended backbone type structures. W. H. James, III, C W. Müller, E. G. Buchanan, M. G. D. Nix, L. Guo, L. Roskop, M. S. Gordon, L. V. Slipchenko, S. H. Gellman, and T. S. Zwier, J. Am. Chem. Soc., 2009, 131(40), 14243-14245.

  6. Measurement and Characterization of Hydrogen-Deuterium Exchange Chemistry Using Relaxation Dispersion NMR Spectroscopy.

    Science.gov (United States)

    Khirich, Gennady; Holliday, Michael J; Lin, Jasper C; Nandy, Aditya

    2018-03-01

    One-dimensional heteronuclear relaxation dispersion NMR spectroscopy at 13 C natural abundance successfully characterized the dynamics of the hydrogen-deuterium exchange reaction occurring at the N ε position in l-arginine by monitoring C δ in varying amounts of D 2 O. A small equilibrium isotope effect was observed and quantified, corresponding to ΔG = -0.14 kcal mol -1 . A bimolecular rate constant of k D = 5.1 × 10 9 s -1 M -1 was determined from the pH*-dependence of k ex (where pH* is the direct electrode reading of pH in 10% D 2 O and k ex is the nuclear spin exchange rate constant), consistent with diffusion-controlled kinetics. The measurement of ΔG serves to bridge the millisecond time scale lifetimes of the detectable positively charged arginine species with the nanosecond time scale lifetime of the nonobservable low-populated neutral arginine intermediate species, thus allowing for characterization of the equilibrium lifetimes of the various arginine species in solution as a function of fractional solvent deuterium content. Despite the system being in fast exchange on the chemical shift time scale, the magnitude of the secondary isotope shift due to the exchange reaction at N ε was accurately measured to be 0.12 ppm directly from curve-fitting D 2 O-dependent dispersion data collected at a single static field strength. These results indicate that relaxation dispersion NMR spectroscopy is a robust and general method for studying base-catalyzed hydrogen-deuterium exchange chemistry at equilibrium.

  7. Modification of interlayer exchange coupling in Fe/V/Fe trilayers using hydrogen

    Energy Technology Data Exchange (ETDEWEB)

    Skoryna, J., E-mail: jskoryna@ifmpan.poznan.pl [Institute of Molecular Physics, Polish Academy of Sciences, Smoluchowskiego 17 St., 60-179 Poznań (Poland); Marczyńska, A. [Institute of Molecular Physics, Polish Academy of Sciences, Smoluchowskiego 17 St., 60-179 Poznań (Poland); Lewandowski, M. [NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85 St., 61-614 Poznań (Poland); Smardz, L. [Institute of Molecular Physics, Polish Academy of Sciences, Smoluchowskiego 17 St., 60-179 Poznań (Poland)

    2015-10-05

    Highlights: • Magnetic films and multilayers. • Thin films. • Hydrogen absorbing materials. • Magnetic measurements. • Exchange coupling. - Abstract: Fe/V/Fe trilayers with constant-thickness Fe and step-like wedged V sublayers were prepared at room temperature using UHV magnetron sputtering. The bottom Fe layer grows onto oxidised Si(1 0 0) substrate and shows relatively high coercivity. The top Fe layer grows on vanadium spacer and shows considerably lower coercivity. The planar growth of the Fe and V sublayers was confirmed in-situ by X-ray photoelectron spectroscopy. Results show that the Fe sublayers are weakly exchange coupled for d{sub V} > 1.4 nm. Results on the coercivity studies as a function of the V interlayer thickness show near d{sub V} ∼ 1.95 nm (∼2.45 nm) weak antiferromagnetic (ferromagnetic) coupling, respectively. The hydrogenation of the Fe/V/Fe trilayers leads to increase of the strength of the ferromagnetic interlayer exchange coupling.

  8. Deuterium exchange with the surface hydrogen of zeolite catalysts. 7. Nickel-containing zeolites

    International Nuclear Information System (INIS)

    Minachev, Kh.M.; Dmitriev, R.V.; Penchev, V.; Kanazirev, V.; Minchev, Kh.; Kasimov, Ch.K.

    1982-01-01

    An in-depth study of heteromolecular isotopic hydrogen exchange (HIHE) in Ni zeolites was undertaken with a view to measuring surface OH group concentrations and determining effectiveness of Ni, on the one hand, and Pd and Pt, on the other, in promoting chemical reactions. Here the degree of metal dispersion in the Ni zeolite was characterized through H 2 chemisorption and thermosorption data. A study was made of the action of these zeolites in catalyzing the disproportionation of toluene. The data obtained here have given an understanding of the effect of the metal, the OH-group concentration, and the mutual arrangement of OH groups and Ni atoms on catalyzed toluene reactions. Results indicated that HIHE occurs on reduced nickel-containing zeolite catalysts at temperatures in excess of 100 0 C, and is limited by the rate of transport of activated hydrogen from the metal particles on the support surface. High-temperature oxidation-reduction of the nickel-containing zeolite-leads to the formation of coarse nickel crystals on the external zeolite crystal faces. Also, the reduced NiCaNaY zeolites show high catalytic activity in the toluene disproportionation only when the nickel has been introduced through ion exchange. Both isotopic exchange and toluene disproportionation are promoted when the nickel particles and OH groups are in close proximity

  9. Gas-phase ion/molecule isotope-exchange reactions: methodology for counting hydrogen atoms in specific organic structural environments by chemical ionization mass spectrometry

    International Nuclear Information System (INIS)

    Hunt, D.F.; Sethi, S.K.

    1980-01-01

    Ion/molecule reactions are described which facilitate exchange of hydrogens for deuteriums in a variety of different chemical environments. Aromatic hydrogens in alkylbenzenes, oxygenated benzenes, m-toluidine, m-phenylenediamine, thiophene, and several polycyclic aromatic hydrocarbons and metallocenes are exchanged under positive ion CI conditions by using either D 2 O, EtOD, or ND 3 as the reagent gas. Aromatic hydrogens, benzylic hydrogens, and hydrogens on carbon adjacent to carbonyl groups suffer exchange under negative ion CI conditions in ND 3 , D 2 O, and EtOD, respectively. A possible mechanism for the exchange process is discussed. 1 figure, 2 tables

  10. Steroid and Protein Ligand Binding to Cytochrome P450 46A1 as Assessed by Hydrogen – Deuterium Exchange and Mass Spectrometry

    Science.gov (United States)

    Liao, Wei-Li; Dodder, Nathan G.; Mast, Natalia; Pikuleva, Irina A.; Turko, Illarion V.

    2009-01-01

    Cytochrome P450 46A1 (CYP46A1) is a key enzyme responsible for cholesterol elimination from the brain. This P450 can interact with different steroid substrates and protein redox partners. We utilized hydrogen-deuterium (H-D) exchange mass spectrometry for investigating CYP46A1-ligand interactions. First, we tested the applicability of the H-D exchange methodology and assessed the amide proton exchange in substrate-free and cholesterol sulfate-bound P450. The results showed good correspondence to the available crystal structures and prompted investigation of the CYP46A1 interactions with the two steroid substrates cholesterol and 24S-hydroxycholesterol and the protein redox partner adrenodoxin (Adx). Compared to substrate-free P450, four peptides in cholesterol-bound CYP46A1 (65–80, 109–116, 151–164, and 351–361) and eight peptides in 24S-hydroxycholesterol-bound enzyme (50–64, 65–80, 109–116, 117–125, 129–143, 151–164, 260–270, and 364–373) showed altered deuterium incorporation. Most of these peptides constitute the enzyme active site, whereas the 351–361 peptide is from the region putatively interacting with the redox partner Adx. This also defines the proximal (presumably water) channel that opens in CYP46A1 upon substrate binding. Reciprocal studies of Adx binding to substrate-free and cholesterol sulfate-bound CYP46A1 revealed changes in the deuteration of the Adx-binding site 144–150 and 351–361 peptides, active site 225–239 and 301–313 peptides, and in the 265–276 peptide, whose functional role is not yet known. The data obtained provide structural insights into how substrate and redox partner binding are coordinated and linked to the hydration of the enzyme active site. PMID:19317426

  11. OPTIMIZATION OF INTERNAL HEAT EXCHANGERS FOR HYDROGEN STORAGE TANKS UTILIZING METAL HYDRIDES

    Energy Technology Data Exchange (ETDEWEB)

    Garrison, S.; Tamburello, D.; Hardy, B.; Anton, D.; Gorbounov, M.; Cognale, C.; van Hassel, B.; Mosher, D.

    2011-07-14

    Two detailed, unit-cell models, a transverse fin design and a longitudinal fin design, of a combined hydride bed and heat exchanger are developed in COMSOL{reg_sign} Multiphysics incorporating and accounting for heat transfer and reaction kinetic limitations. MatLab{reg_sign} scripts for autonomous model generation are developed and incorporated into (1) a grid-based and (2) a systematic optimization routine based on the Nelder-Mead downhill simplex method to determine the geometrical parameters that lead to the optimal structure for each fin design that maximizes the hydrogen stored within the hydride. The optimal designs for both the transverse and longitudinal fin designs point toward closely-spaced, small cooling fluid tubes. Under the hydrogen feed conditions studied (50 bar), a 25 times improvement or better in the hydrogen storage kinetics will be required to simultaneously meet the Department of Energy technical targets for gravimetric capacity and fill time. These models and methodology can be rapidly applied to other hydrogen storage materials, such as other metal hydrides or to cryoadsorbents, in future work.

  12. Hydrogen-deuterium exchange studies utilizing a thermospray mass spectrometer interface

    International Nuclear Information System (INIS)

    Siegel, M.M.

    1988-01-01

    A routine and efficient technique was developed for exchanging acidic hydrogen for deuterium, utilizing a thermospray mass spectrometer interface, to produce deuterium-enriched deuteron-adduct molecular and fragment ions. Samples dissolved in solvents containing acidic deuterium atoms (D 2 O/trifluoroacetic acid-d 1 or ammonium acetate-d 4 ) were introduced via slug injection into the thermospray interface sandwiched between slugs of the deuteriated solvent. At all other times, the carrier solvent system introduced into the mass spectrometer consisted of a non-deuterium-enriched mixture. Deuterium enrichments achieved were greater than 95%. Deuterium-enriched deuteron-adduct molecular and fragment ions enabled the determination of the number of acidic hydrogens and aided in structural elucidation of model compounds of pharmaceutical interest

  13. AMORE-HX: a multidimensional optimization of radial enhanced NMR-sampled hydrogen exchange

    International Nuclear Information System (INIS)

    Gledhill, John M.; Walters, Benjamin T.; Wand, A. Joshua

    2009-01-01

    The Cartesian sampled three-dimensional HNCO experiment is inherently limited in time resolution and sensitivity for the real time measurement of protein hydrogen exchange. This is largely overcome by use of the radial HNCO experiment that employs the use of optimized sampling angles. The significant practical limitation presented by use of three-dimensional data is the large data storage and processing requirements necessary and is largely overcome by taking advantage of the inherent capabilities of the 2D-FT to process selective frequency space without artifact or limitation. Decomposition of angle spectra into positive and negative ridge components provides increased resolution and allows statistical averaging of intensity and therefore increased precision. Strategies for averaging ridge cross sections within and between angle spectra are developed to allow further statistical approaches for increasing the precision of measured hydrogen occupancy. Intensity artifacts potentially introduced by over-pulsing are effectively eliminated by use of the BEST approach

  14. Thermodynamics of hydrogen adsorption on calcium-exchanged faujasite-type zeolites

    Energy Technology Data Exchange (ETDEWEB)

    Palomino, G.T.; Arean, C.O.; Carayol, M.R.L. [Departamento de Quimica, Universidad de las Islas Baleares, 07122 Palma de Mallorca (Spain); Bonelli, B.; Armandi, M.; Garrone, E. (Dipartimento di Scienza dei Materiali ed Ingegneria Chimica, Politecnico di Torino, 10129 Turin, Italy, and INSTM Unit of Torino Politecnico); Parra, J.B.; Ania, C.O. [CSIC, Instituto Nacional del Carbon, Apdo. 73, E-33080 Oviedo (Spain)

    2009-05-15

    A combination of variable-temperature infrared spectroscopy with volumetric gas adsorption measurements was used to study the thermodynamics of hydrogen adsorption, at a low temperature, on calcium-exchanged zeolites X and Y. Two adsorption regimes were considered: (i) localized adsorption of dihydrogen molecules on Ca{sup 2+} cation sites, and (ii) delocalized hydrogen adsorption following saturation of the Ca{sup 2+} adsorbing centres. For localized adsorption, the corresponding enthalpy change was found to be in the range of -12 to -15 kJ mol{sup -1}, while the isosteric heat of delocalized adsorption was found to be in the range of 4.5-5.5 kJ mol{sup -1}. These experimental results are discussed in the broader context of corresponding data for other alkaline zeolites, with a focus on correlation between adsorption enthalpy and entropy for the localized adsorption regime. (author)

  15. Electrochemistry Modeling of Proton Exchange Membrane (PEM) Water Electrolysis for Hydrogen Production

    International Nuclear Information System (INIS)

    Meng Ni; Michael KH Leung; Dennis YC Leung

    2006-01-01

    An electrochemistry model was developed to analyse the J-V characteristics of a Proton Exchange Membrane (PEM) water electrolyzer for hydrogen production. The Butler-Volmer equation and water transport characteristics through electrolyte membrane were employed to simulate the electrode activation over-potential and membrane ohmic over-potential, respectively. The modeling results are found to agree reasonably well with experimental data published in the literature. The parametric simulations show that the ohmic over-potential is relatively small with typical water content in the membrane. Compared with the cathode over-potential, the anode over-potential is more significant and constitutes the major source of voltage loss. The high anode over-potential is due to the relatively slow oxidation kinetics, which is related to anode material property and microstructure. This model can be integrated with a photovoltaic or wind turbine model to predict the performance of sustainable hydrogen production systems and optimise their designs. (authors)

  16. NMR-based detection of hydrogen/deuterium exchange in liposome-embedded membrane proteins.

    Directory of Open Access Journals (Sweden)

    Xuejun Yao

    Full Text Available Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.

  17. Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry

    Directory of Open Access Journals (Sweden)

    Tuma Roman

    2008-04-01

    Full Text Available Abstract Recent advances in protein mass spectrometry (MS have enabled determinations of hydrogen deuterium exchange (HDX in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

  18. High-sensitivity mass spectrometry for imaging subunit interactions: hydrogen/deuterium exchange.

    Science.gov (United States)

    Lanman, Jason; Prevelige, Peter E

    2004-04-01

    In recent years, advances in mass spectrometry have provided unprecedented knowledge of protein expression within cells. It has become apparent that many proteins function as macromolecular complexes. Structural genomics programs are determining the fold of these proteins at an increasing rate and electron microscopic tomography potentially provides a means to determine the location of these complexes within the cell. A complete understanding of the molecular mechanism of these proteins requires detailed information on the interactions and dynamics within the complex. Recent advances in mass spectrometry now make it possible to use hydrogen/deuterium exchange to detect intersubunit interfaces and dynamics within supramolecular complexes.

  19. Selection of the process for the heavy water production using isotopic exchange amonia-hydrogen

    International Nuclear Information System (INIS)

    Guzman R, G.H.

    1980-01-01

    The utilization of the Petroleos Mexicanos ammonia plants for heavy water production by the isotopic exchange NH 3 -H 2 process is presented, in addition a description of the other heavy water production processes was presented. In the ammonia hydrogen process exist two possible alternatives for the operation of the system, one of them is to carry out the enrichment to the same temperature, the second consists in making the enrichment at two different temperatures (dual temperature process), an analysis was made to select the best alternative. The conclusion was that the best operation is the dual temperature process, which presents higher advantages according to the thermodynamics and engineering of the process. (author)

  20. Application of solid state catalytic hydrogen isotope exchange to the tritium labeling of lyoszyme

    International Nuclear Information System (INIS)

    Filikov, A.V.; Jones, J.R.

    1995-01-01

    Solid state catalytic hydrogen isotope exchange has been employed to label hen egg lysozyme with tritium. Optimization of reaction conditions so that amino acids and peptide bonds remained intact led to a tritiated products with 97% of the original enzymatic activity and 94% radiochemical purity. The specific activity when using a T 2 :H 2 mixture of 1:1000, was 16 mCi·mmol -1 . It is suggested that the currently adopted approach may have wide applications for other proteins able to tolerate lyophilization conditions without loss of activity. (Author)

  1. Aspartic protease nepenthesin-1 as a tool for digestion in hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Kadek, Alan; Mrazek, Hynek; Halada, Petr; Rey, Martial; Schriemer, David C; Man, Petr

    2014-05-06

    Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) utilizes enzymatic digestion of proteins to localize the information about altered exchange patterns in protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. Recently, it was shown that there is an interesting group of proteolytic enzymes from carnivorous pitcher plants of the genus Nepenthes. In this report, we describe successful immobilization and the use of one of these enzymes, nepenthesin-1, in HXMS workflow. In contrast to pepsin, it has different cleavage specificities, and despite its high inherent susceptibility to reducing and denaturing agents, it is very stable upon immobilization and withstands even high concentration of guanidine hydrochloride and reducing agents. We show that denaturing agents can alter digestion by reducing protease activity and/or substrate solubility, and additionally, they influence the trapping of proteolytic peptides onto the reversed phase resin.

  2. Hydrogen production in the K-Basin ion exchange columns, modules and cartridge filters

    Energy Technology Data Exchange (ETDEWEB)

    1994-12-21

    K-Basin uses ion exchange modules and ion exchange (IX) columns for removing radionuclides from the basin water. When the columns and modules are loaded, they are removed from service, drained and stored. After a few IX columns accumulate in storage, they are moved to a burial box. One of the burial box contains 33 columns and the other, six. The radionuclides act on the liquid left within and adhering to the beads to produce hydrogen. This report describes the generation rate, accumulation rate and significance of that accumulation. This summary also highlights those major areas of concern to the external (to Westinghouse Hanford Company [WHC]) reviewers. Appendix H presents the comments made by the external reviewers and, on a separate sheet, the responses to those comments. The concerns regarding the details of the analytical approach, are addressed in Appendix H and in the appropriate section.

  3. Deuterium exchange between hydrogen and water in a trickle bed reactor

    International Nuclear Information System (INIS)

    Enright, J.T.; Chuang, T.T.

    1978-01-01

    The catalyzed exchange of deuterium between hydrogen and liquid water has been studied as the basis for a heavy water production process. Platinum catalyst which had been waterproofed with Teflon was tested in a 0.2 m diameter trickle bed reactor at pressures and temperatures up to 6 MPa and 440 K. Extensive experimental data were used to test a model of the system which was developed from fundamental principles. It was found that mass transfer plays a very important role in the overall exchange and the conventional theory of vapour/liquid mass transfer does not adequately describe the absorption process. Modelling of the data has resulted in the postulation of a second method of mass transfer whereby HDO transfers directly from the catalyst to the bulk liquid phase. (author)

  4. Oligomerization interface of RAGE receptor revealed by MS-monitored hydrogen deuterium exchange.

    Directory of Open Access Journals (Sweden)

    Ewa Sitkiewicz

    Full Text Available Activation of the receptor for advanced glycation end products (RAGE leads to a chronic proinflammatory signal, affecting patients with a variety of diseases. Potentially beneficial modification of RAGE activity requires understanding the signal transduction mechanism at the molecular level. The ligand binding domain is structurally uncoupled from the cytoplasmic domain, suggesting receptor oligomerization is a requirement for receptor activation. In this study, we used hydrogen-deuterium exchange and mass spectrometry to map structural differences between the monomeric and oligomeric forms of RAGE. Our results indicated the presence of a region shielded from exchange in the oligomeric form of RAGE and led to the identification of a new oligomerization interface localized at the linker region between domains C1 and C2. Based on this finding, a model of a RAGE dimer and higher oligomeric state was constructed.

  5. Oligomerization interface of RAGE receptor revealed by MS-monitored hydrogen deuterium exchange.

    Science.gov (United States)

    Sitkiewicz, Ewa; Tarnowski, Krzysztof; Poznański, Jarosław; Kulma, Magdalena; Dadlez, Michal

    2013-01-01

    Activation of the receptor for advanced glycation end products (RAGE) leads to a chronic proinflammatory signal, affecting patients with a variety of diseases. Potentially beneficial modification of RAGE activity requires understanding the signal transduction mechanism at the molecular level. The ligand binding domain is structurally uncoupled from the cytoplasmic domain, suggesting receptor oligomerization is a requirement for receptor activation. In this study, we used hydrogen-deuterium exchange and mass spectrometry to map structural differences between the monomeric and oligomeric forms of RAGE. Our results indicated the presence of a region shielded from exchange in the oligomeric form of RAGE and led to the identification of a new oligomerization interface localized at the linker region between domains C1 and C2. Based on this finding, a model of a RAGE dimer and higher oligomeric state was constructed.

  6. Hydrogen production in the K-Basin ion exchange columns, modules and cartridge filters

    International Nuclear Information System (INIS)

    1994-01-01

    K-Basin uses ion exchange modules and ion exchange (IX) columns for removing radionuclides from the basin water. When the columns and modules are loaded, they are removed from service, drained and stored. After a few IX columns accumulate in storage, they are moved to a burial box. One of the burial box contains 33 columns and the other, six. The radionuclides act on the liquid left within and adhering to the beads to produce hydrogen. This report describes the generation rate, accumulation rate and significance of that accumulation. This summary also highlights those major areas of concern to the external (to Westinghouse Hanford Company [WHC]) reviewers. Appendix H presents the comments made by the external reviewers and, on a separate sheet, the responses to those comments. The concerns regarding the details of the analytical approach, are addressed in Appendix H and in the appropriate section

  7. HDXFinder: Automated Analysis and Data Reporting of Deuterium/Hydrogen Exchange Mass Spectrometry

    Science.gov (United States)

    Miller, Danny E.; Prasannan, Charulata B.; Villar, Maria T.; Fenton, Aron W.; Artigues, Antonio

    2012-02-01

    Hydrogen/deuterium exchange in combination with mass spectrometry (H/D MS) is a sensitive technique for detection of changes in protein conformation and dynamics. However, wide application of H/D MS has been hindered, in part, by the lack of computational tools necessary for efficient analysis of the large data sets associated with this technique. We report a novel web-based application for automatic analysis of H/D MS experimental data. This application relies on the high resolution of mass spectrometers to extract all isotopic envelopes before correlating these envelopes with individual peptides. Although a fully automatic analysis is possible, a variety of graphical tools are included to aid in the verification of correlations and rankings of the isotopic peptide envelopes. As a demonstration, the rate constants for H/D exchange of peptides from rabbit muscle pyruvate kinase are mapped onto the structure of this protein.

  8. Hydrogen/Deuterium Exchange Study of Subtilisin Carlsberg During Prolonged Exposure to Organic Solvents

    Science.gov (United States)

    Fasoli, Ezio; Ferrer, Amaris; Barletta, Gabriel L.

    2009-01-01

    It has been previously reported that prolonged exposure of an enzyme to organic solvents leads to substantial decrease of activity. This effect was found to be unrelated to the catalysts’ structure or their possible aggregation in organic solvents, and up to the present day the cause for activity loss remains unclear. In the present work, the structural dynamics of the serine protease subtilisin Carlsberg (SC) have been investigated during prolonged exposure to two organic solvents by following hydrogen/deuterium (H/D) exchange of mobile protons. The enzyme, after lyophilization, was incubated in organic solvents at controlled deuteriated water activity for different times and the H/D exchange was allowed to take place. The amount of deuterium exchanged was evaluated by 2H NMR, which in turn gave us a picture of the changing dynamics of our model enzyme during incubation and under different experimental conditions. Our results show that the flexibility of SC decreases during prolonged storage in 1,4-dioxane (Diox) and acetonitrile (ACN) as indicated by the observed 3- to 10-fold decrease in the apparent rate constants of exchange (k) of fast exchangeable protons (FEP) and slow exchangeable protons (SEP) in the protein. Our study also shows that SC is more flexible in ACN than in Diox (k 3−20 times higher in ACN for the FEP and SEP), suggesting that enzyme dynamics are affected by solvent physicochemical properties. Additionally, the enzyme dynamics are also affected by the method of preparation: decreased flexibility (k decreases 3- to 10-fold for FEP and SEP) is observed when the enzyme is chemically modified with poly ethylene glycol (PEGylated) or colyophilized with crown ethers. A possible relationship between activity, enantioselectivity (E), and structural dynamics is discussed, demonstrating that direct correlations, as have been attempted in the past, are hampered by the multi-variable nature and complexity of the system. PMID:18985614

  9. Microsecond pulsed hydrogen/deuterium exchange of electrosprayed ubiquitin ions stored in a linear ion trap.

    Science.gov (United States)

    Rajabi, Khadijeh

    2015-02-07

    A pulse of D2O vapour on the order of microseconds is allowed to react with the +6 to +9 charge states of ubiquitin confined in a linear ion trap (LIT). Two envelopes of peaks are detected for the ions of ubiquitin, corresponding to the ions that exchange more quickly and more slowly. The deuterium uptake of the protonated sites on ubiquitin ions accounts for the ion population with the fast exchange. The hydrogen/deuterium exchange (HDX) kinetics of ubiquitin ions trapped in the LIT for 200 ms showed comparable structural transitions to those trapped for 300 ms. When ions are trapped for longer, i.e. up to 2000 ms, mainly the slow exchanging ion population is detected. In all experiments the +7 ions exchange the most, suggesting a short distance between the surface protonated sites and nearby charged sites, and concomitantly high accessibility of surface protonated sites towards D2O. The +6 ions are more compact than the +7 ions but have one fewer protonated site, therefore fewer surface availabilities for D2O attack. The data suggest that the +6 ions keep most of their solution-phase contacts intact while the hydrophobic core is slightly interrupted in the +7 ions, possibly due to the exposure of charged His68 that is normally buried in the hydrophobic pocket. The +8 and +9 ions have more protonated sites but are less compact than the +7 ions because of Coulombic repulsion, resulting in a larger distance between the protonated sites and the basic sites. The data indicate that the HDX mechanism of ions with the slower exchange corresponding to the second envelope of peaks is primarily governed via a relay mechanism. The results suggest that the pulsed HDX MS method is sampling a population of ubiquitin ions with a similar backbone fold to the solution.

  10. Spectroscopic studies on hydrogen/deuterium exchange equilibria and related phenomena in aqueous solution

    International Nuclear Information System (INIS)

    Taylor, C.E.

    1978-07-01

    This thesis describes spectroscopic studies on the aqueous phase hydroxide ion and acid dissociation of the 3-cyano pyridinium ion. Firstly, NMR spectroscopy was used, in the fast exchange mode, to measure the hydrogen isotope fractionation constants for the exchange sites in the hydroxide ion and its solvation shell. As part of the above analysis, values have been derived for the NMR chemical shifts of the hydroxide ion and solvation shell protons. Secondly, the NMR fast exchange method was used to assess the effect of temperature on the hydroxide ion fractionation constant. Thirdly, the NMR fast exchange method was used to assess the nature and structure of ion pairs of the type M + , OH - (H 2 O)sub(p) where M = Tl, Na, K, Li. Data necessary to these analyses were the Ion Chemical Shift of the thallous ion and the volume magnetic susceptibilities of some MOH solutions. These have been measured and are reported here, the latter being determined over a wide concentration range. Finally, U.V. - Visible spectroscopy was employed to measure the pKsub(H) of the 3-cyano pyridinium ion over a range of temperatures and the pKsub(D) at 298.5 K. The results are given and discussed. (U.K.)

  11. Hydrogen-deuterium exchange in spiro cyclic ketones: a search for intramolecular catalysis

    International Nuclear Information System (INIS)

    Hsu, R.C.L.

    1979-01-01

    The synthesis and characterization of 1'-hydroxy-3',3'-dimethylspiro[cyclohexane-1,2'-indan]-4-one (17b), 1'-amino-3',3'-dimethylspiro[cyclohexane-1,2'-indan]4-one (6a), 1'-N,N-dimethylamino-3',3'-dimethylspiro[cyclohexane-1,2'-indan]-4-one (6c) and 1'-[N-ethyl-α(d 2 )-amino]-3'-3'-dimethylspiro[cyclohexane-1,2'-indan]-4-one (6b) are described. The hydrogen-deuterium exchange reactions of aminoketones 6b and 6c with CH 3 OD are too fast to be followed by the mass spectrometric methodology. However, the H/D exchange between hydroxyketone 17b and CH 3 OD is shown to be intramolecularly catalyzed under both basic and neutral conditions. The rate constant for the exchange of C(3)-axial-H in 17b is 35 times greater than the rate constant for the exchange of an axial α-H in 4-t-butylcyclohexanone

  12. Proceedings of the fourth information exchange meeting on nuclear production of hydrogen

    International Nuclear Information System (INIS)

    2010-01-01

    The use of hydrogen, both as feedstock for the industry (oil and chemical) and as an energy carrier, is expected to grow substantially during the coming decades. The current predominant method of producing hydrogen by steam-reforming methane (from natural gas) is not sustainable and has environmental drawbacks, including the emission of greenhouse gasses (GHGs). Nuclear energy offers a way to produce hydrogen from water without depleting natural gas, a valuable natural resource, and without the emission of GHGs. The OECD Nuclear Energy Agency (NEA) has conducted a number of information exchange meetings with the objective of stimulating progress in the development of nuclear production of hydrogen. These meetings, held in 2000 in Paris, France, in 2003 in Argonne, Illinois, USA, and in 2005 in Oarai, Japan, were well-attended and very successful. It is hoped that the information presented at fourth meeting and contained in these proceedings may be useful in advancing the objective of achieving economically viable, sustainable and emission-free production of hydrogen. The need for a sustainable supply of clean energy is one of the main problems facing the world. Among the various energy technologies which may be considered (including hydro, wind, solar, geo-thermal, wave and tidal), only nuclear - through the use of fast-neutron fission reactors - is capable of delivering the copious quantities of sustainable energy that will be required. In view of this, one of the means under consideration for achieving the objective of nuclear-produced hydrogen is enhanced international cooperation, including the establishment of one or more OECD/NEA joint projects. In this respect, it is worth noting that similar joint projects undertaken in the past (for example, the Dragon Project and the Halden Reactor Project) have been highly beneficial and have provided significant amounts of useful information to the sponsoring countries at shared costs. This report describes the

  13. Enumeration of labile hydrogens in natural organic matter by use of hydrogen/deuterium exchange Fourier transform ion cyclotron resonance mass spectrometry.

    Science.gov (United States)

    Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Kharybin, Oleg; Perminova, Irina; Konstantinov, Andrey; Nikolaev, Eugene

    2013-11-19

    A method to enumerate labile hydrogens in all constituents of molecular ensemble of natural organic matter (NOM) based on our previously developed simple hydrogen/deuterium (H/D) exchange (electrospray ionization (ESI) ion source (Kostyukevich et al. Anal. Chem. 2013, 85, 5330) and ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry is presented. The method was applied for analysis of Suwannee River fulvic acid (SRFA), which is an International Humic Substances Society standard, as well as Siberian crude oil; and lignosulfonate. We found that SRFA and lignosulfonate molecules contain 2-5 labile hydrogens, and their number increases with the number of oxygens in the molecule. Also, we observed that compounds of Siberian crude oil ionizing in positive-ESI mode do not have labile hydrogens, while compounds ionizing in negative-ESI mode have one labile hydrogen that detaches during ESI ionization.

  14. Synthesis, morphology, and properties of segmented poly(ether amide)s with uniform oxalamide-based hard segments

    NARCIS (Netherlands)

    Sijbrandi, N.J.; Kimenai, A.J.; Mes, E.P.C.; Broos, R.; Bar, G.; Rosenthal, M.; Odarchenko, Y.; Ivanov, D.A.; Dijkstra, Pieter J.; Feijen, Jan

    2012-01-01

    The synthesis, morphology, and properties of segmented poly(ether amide)s based on flexible PTHF segments (Mn = 1.1 × 103 g mol–1) and uniform rigid oxalamide segments were investigated. The amount of oxalamide groups in the hard segment and the spacer length of bisoxalamide-based hydrogen bonded

  15. Probing conserved helical modules of portal complexes by mass spectrometry-based hydrogen/deuterium exchange.

    Science.gov (United States)

    Kang, Sebyung; Poliakov, Anton; Sexton, Jennifer; Renfrow, Matthew B; Prevelige, Peter E

    2008-09-05

    The Double-stranded DNA bacteriophage P22 has a ring-shaped dodecameric complex composed of the 84 kDa portal protein subunit that forms the central channel of the phage DNA packaging motor. The overall morphology of the P22 portal complex is similar to that of the portal complexes of Phi29, SPP1, T3, T7 phages and herpes simplex virus. Secondary structure prediction of P22 portal protein and its threading onto the crystal structure of the Phi29 portal complexes suggested that the P22 portal protein complex shares conserved helical modules that were found in the dodecameric interfaces of the Phi29 portal complex. To identify the amino acids involved in intersubunit contacts in the P22 portal ring complexes and validate the threading model, we performed comparative hydrogen/deuterium exchange analysis of monomeric and in vitro assembled portal proteins of P22 and the dodecameric Phi29 portal. Hydrogen/deuterium exchange experiments provided evidence of intersubunit interactions in the P22 portal complex similar to those in the Phi29 portal that map to the regions predicted to be conserved helical modules.

  16. Probing Conserved Helical Modules of Portal Complexes by Mass Spectrometry based Hydrogen/deuterium Exchange

    Science.gov (United States)

    Kang, Sebyung; Poliakov, Anton; Sexton, Jennifer; Renfrow, Matthew B.; Prevelige, Peter E.

    2008-01-01

    The dsDNA bacteriophage P22 has a ring shaped dodecameric complex composed of the 84 kDa portal protein subunit which forms the central channel of the phage’s DNA packaging motor. The overall morphology of the P22 portal complex is similar to that of the portal complexes of Phi29, SPP1, T3, T7 phages and herpes simplex virus. Secondary structure prediction of P22 portal protein and its threading onto the crystal structure of the Phi29 portal complexes suggested that P22 portal protein complex shares conserved helical modules which were found in the dodecameric interfaces of the Phi29 portal complex. To identify the amino acids involved in inter-subunit contacts in the P22 portal ring complexes and validate the threading model, we performed comparative hydrogen/deuterium exchange analysis of monomeric and in vitro assembled portal proteins of P22 and the dodecameric Phi29 portal. Hydrogen/deuterium exchange experiments provided evidence of inter-subunit interactions in the P22 portal complex similar to those in the Phi29 portal which map to the regions predicted to be conserved helical modules. PMID:18621389

  17. Pulsed hydrogen-deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation.

    Science.gov (United States)

    Zhang, Ying; Rempel, Don L; Zhang, Jun; Sharma, Anuj K; Mirica, Liviu M; Gross, Michael L

    2013-09-03

    Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aβ42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu(2+) ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.

  18. Hydrogen Temperature-Programmed Desorption in Platinum Catalysts: Decomposition and Isotopic Exchange by Spillover Hydrogen of Chemisorbed Ammonia.

    NARCIS (Netherlands)

    Koningsberger, D.C.; Miller, J.T.; Meyers, B.L.; Barr, M.K.; Modica, F.S.

    1996-01-01

    H{2}-TPD of Pt/alumina catalysts display multiple hydrogendesorptions. In addition to chemisorbed hydrogen (Peak I) atapproximately 175}o{C, there is a small hydrogen desorption (PeakII) at about 250}o{C and a large, irreversible hydrogen desorption(Peak III) at 450}o{C. The quantity of hydrogen

  19. Method of making hydrophobic industrial catalyst for water-hydrogen isotope exchange

    International Nuclear Information System (INIS)

    Sato, Toshio; Ookoshi, Sumio; Takahashi, Tomiki

    1982-01-01

    The authors have performed the research centering around the development of platinum catalyst carried by styrene divinylbenzene copolymer as the hydrophobic catalyst for water-hydrogen isotope exchange for the purpose of heavy water concentration and especially tritium removal. In this paper, the method of industrial production of this catalyst, the results of catalytic performance test by trickle bed and the problems are reported. It was found that only chloroplatinic acid was suitable as the practical raw material of the catalyst. The ethanol solution of chloroplatinic acid is practically most desirable. Generally, the catalytic activity increases by the aging of SDB in pure hydrogen flow. For the impregnation of chloroplatinic acid into SDB, the column method is suitable. The impregnated carriers are dried with an air drier. Then the carriers carrying chloroplatinic acid are reduced in a reaction tube with highly pure hydrogen. The catalytic performance test was performed in a packed tower, and the effects of the shape of catalysts, flow mode, oxygen, scale-up, pressure and impurities are reported. (Kako, I.)

  20. Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus.

    Science.gov (United States)

    Gu, Zhenyu; Zitzewitz, Jill A; Matthews, C Robert

    2007-04-27

    To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good

  1. Electrostatic stabilization and general base catalysis in the active site of the human protein disulfide isomerase a domain monitored by hydrogen exchange.

    Science.gov (United States)

    Hernández, Griselda; Anderson, Janet S; LeMaster, David M

    2008-03-25

    The nucleophilic Cys36 thiol of the human protein disulfide isomerase a domain is positioned over the N terminus of the alpha(2) helix. Amides in the active site exhibit diffusion-limited, hydroxide-catalyzed exchange, indicating that the local positive electrostatic potential decreases the pK value for peptide anion formation by at least 2 units so as to equal or exceed the acidity of water. In stark contrast to the pH dependence of exchange for simple peptides, the His38 amide in the reduced enzyme exhibits a maximum rate of exchange at pH 5 due to efficient general base catalysis by the neutral imidazole of its own side chain and suppression of its exchange by the ionization of the Cys36 thiol. Ionization of this thiol and deprotonation of the His38 side chain suppress the Cys39 amide hydroxide-catalyzed exchange by a million-fold. The electrostatic potential within the active site monitored by these exchange experiments provides a means of stabilizing the two distinct transition states that lead to substrate reduction and oxidation. Molecular modeling offers a role for the conserved Arg103 in coordinating the oxidative transition-state complex, thus providing further support for mechanisms of disulfide isomerization that utilize enzymatic catalysis at each step of the overall reaction.

  2. A study of isotopic exchange of hydrogen and deuterium in a LaNi3Al2 hydride bed

    International Nuclear Information System (INIS)

    Fukada, S.; Matsuo, H.; Okunaga, T.; Mitsuishi, N.

    1992-01-01

    An experimental study was carried out on the isotopic exhange of hydrogen and deuterium in a LaNi 3 Al 2 hydride bed. The exchange capacity of the LaNi 3 Al 2 hydride for deuterium, the overall mass-transfer coefficient and the height equivalent to a theoretical plate (HETP) were obtained from the analysis of effluent curves for a pulse or step change of an influent deuterium concentration. The exchange capacity for deuterium was found to be almost constant between 200 and 300deg C and was around 1.7 in a D/metal ratio. The rate of the isotopic exchange was independent of the total hydrogen pressure, and a value of 31.8 kJ/mol was obtained for its activation energy. The minimum HETP value obtained was 0.5 cm at 300deg C and is smaller than that of a cryogenic distillation column for hydrogen isotopic separation. (orig.)

  3. Combining size-exclusion chromatography with differential hydrogen-deuterium exchange to study protein conformational changes.

    Science.gov (United States)

    Makarov, Alexey A; Helmy, Roy

    2016-01-29

    Methods for protein characterization are being actively developed based on the growing importance of protein therapies and applications. The goal of this study was to demonstrate the use of size-exclusion chromatography (SEC) in combination with differential hydrogen-deuterium exchange (HDX) to compare protein global conformational changes at different solution conditions. Using chaotropic mobile phase additive, differential HDX was used to detect a number of solvent accessible labile protons of protein on-column at pH and temperature conditions which provided unrestricted intrinsic H/D exchange (all-or-nothing approach). Varying SEC on-column conditions allowed for protein conformational changes to be observed. Temperature and pressure were independently studied with regards to their effect on the proteins' (insulin, cytochrome C, ubiquitin, and myoglobin) conformational changes in the solution. The obtained ΔHDX profiles revealed protein conformational changes in solution under varied conditions manifested as the difference in the number of protons exchanged to deuterons, or vice-versa. The approach described in this manuscript could prove useful for protein batch-to-batch comparisons, for optimization of chemical reactions with enzyme as catalyst or for protein chemical modification reactions. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Functional Dynamics of Hexameric Helicase Probed by Hydrogen Exchange and Simulation

    Science.gov (United States)

    Radou, Gaël; Dreyer, Frauke N.; Tuma, Roman; Paci, Emanuele

    2014-01-01

    The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason, it is a significant challenge to investigate these assemblies using conventional experimental techniques. One of the most promising experimental techniques is hydrogen-deuterium exchange detected by mass spectrometry. Here, we describe to our knowledge a new computational method for quantitative interpretation of deuterium exchange kinetics and apply it to a hexameric viral helicase P4 that unwinds and translocates RNA into a virus capsid at the expense of ATP hydrolysis. Room-temperature dynamics probed by a hundred nanoseconds of all-atom molecular dynamics simulations is sufficient to predict the exchange kinetics of most sequence fragments and provide a residue-level interpretation of the low-resolution experimental results. The strategy presented here is also a valuable tool to validate experimental data, e.g., assignments, and to probe mechanisms that cannot be observed by x-ray crystallography, or that occur over timescales longer than those that can be realistically simulated, such as the opening of the hexameric ring. PMID:25140434

  5. Sodium/hydrogen-exchanger inhibition during cardioplegic arrest and cardiopulmonary bypass: an experimental study.

    Science.gov (United States)

    Cox, Charles S; Sauer, Henning; Allen, Steven J; Buja, L Maximilian; Laine, Glen A

    2002-05-01

    We sought to determine whether pretreatment with a sodium/hydrogen-exchange inhibitor (EMD 96 785) improves myocardial performance and reduces myocardial edema after cardioplegic arrest and cardiopulmonary bypass. Anesthetized dogs (n = 13) were instrumented with vascular catheters, myocardial ultrasonic crystals, and left ventricular micromanometers to measure preload recruitable stroke work, maximum rate of pressure rise (positive and negative), and left ventricular end-diastolic volume and pressure. Cardiac output was measured by means of thermodilution. Myocardial tissue water content was determined from sequential biopsy. After baseline measurements, hypothermic (28 degrees C) cardiopulmonary bypass was initiated. Cardioplegic arrest (4 degrees C Bretschneider crystalloid cardioplegic solution) was maintained for 2 hours, followed by reperfusion-rewarming and separation from cardiopulmonary bypass. Preload recruitable stroke work and myocardial tissue water content were measured at 30, 60, and 120 minutes after bypass. EMD 96 785 (3 mg/kg) was given 15 minutes before bypass, and 2 micromol was given in the cardioplegic solution. Control animals received the same volume of saline vehicle. Arterial-coronary sinus lactate difference was similar in both animals receiving EMD 96 785 and control animals, suggesting equivalent myocardial ischemia in each group. Myocardial tissue water content increased from baseline in both animals receiving EMD 96 785 and control animals with cardiopulmonary bypass and cardioplegic arrest but was statistically lower in animals receiving EMD 96 785 compared with control animals (range, 1.0%-1.5% lower in animals receiving EMD 96 785). Preload recruitable stroke work decreased from baseline (97 +/- 2 mm Hg) at 30 (59 +/- 6 mm Hg) and 60 (72 +/- 9 mm Hg) minutes after cardiopulmonary bypass and cardioplegic arrest in control animals; preload recruitable stroke work did not decrease from baseline (98 +/- 2 mm Hg) in animals receiving

  6. Analysis of Hydrogen Isotopic Exchange: Lava Creek Tuff Ash and Isotopically Labeled Water

    Science.gov (United States)

    Ross, A. M.; Seligman, A. N.; Bindeman, I. N.; Nolan, G. S.

    2015-12-01

    Nolan and Bindeman (2013) placed secondarily hydrated ash from the 7.7 ka eruption of Mt. Mazama (δD=-149‰, 2.3wt% H2Ot) in isotopically labeled water (+650 ‰ δD, +56 ‰ δ18O) and observed that the H2Ot and δ18O values remained constant, but the δD values of ash increased with the surrounding water at 20, 40 and 70 °C. We expand on this work by conducting a similar experiment with ash from the 640 ka Lava Creek Tuff (LCT, δD of -128 ‰; 2.1 wt.% H2Ot) eruption of Yellowstone to see if significantly older glass (with a hypothesized gel layer on the surface shielding the interior from alteration) produces the same results. We have experiments running at 70, 24, and 5 °C, and periodically remove ~1.5 mg of glass to measure the δD (‰) and H2Ot (wt.%) of water extracted from the glass on a TC/EA MAT 253 continuous flow system. After 600 hours, the δD of the samples left at 5 and 24 °C remains at -128 ‰, but increased 8‰ for the 70 °C run series. However, there is no measurable change in wt.% of H2Ot, indicating that hydrogen exchange is not dictated by the addition of water. We are measuring and will report further progress of isotope exchange. We also plan to analyze the water in the LCT glass for δ18O (‰) to see if, as is the case for the Mt. Mazama glass, the δ18O (‰) remains constant. We also analyzed Mt. Mazama glass from the Nolan and Bindeman (2013) experiments that have now been sitting in isotopically labeled water at room temperature for ~5 years. The water concentration is still unchanged (2.3 wt.% H2Ot), and the δD of the water in the glass is now -111 ‰, causing an increase of 38 ‰. Our preliminary results show that exchange of hydrogen isotopes of hydrated glass is not limited by the age of the glass, and that the testing of hydrogen isotopes of secondarily hydrated glass, regardless of age, may not be a reliable paleoclimate indicator.

  7. Detection of the sulfhydryl groups in proteins with slow hydrogen exchange rates and determination of their proton/deuteron fractionation factors using the deuterium-induced effects on the 13C(beta) NMR signals.

    Science.gov (United States)

    Takeda, Mitsuhiro; Jee, JunGoo; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-05-05

    A method for identifying cysteine (Cys) residues with sulfhydryl (SH) groups exhibiting slow hydrogen exchange rates has been developed for proteins in aqueous media. The method utilizes the isotope shifts of the C(beta) chemical shifts induced by the deuteration of the SH groups. The 18.2 kDa E. coli peptidyl prolyl cis-trans isomerase b (EPPIb), which was selectively labeled with [3-(13)C;3,3-(2)H(2)]Cys, showed much narrower line widths for the (13)C(beta) NMR signals, as compared to those of the proteins labeled with either [3-(13)C]Cys or (3R)-[3-(13)C;3-(2)H]Cys. The (13)C(beta) signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-(13)C;3,3-(2)H(2)]Cys, split into four signals in H(2)O/D(2)O (1:1) at 40 degrees C and pH 7.5, indicating that the exchange rates of the side-chain SH's and the backbone amides are too slow to average the chemical shift differences of the (13)C(beta) signals, due to the two- and three-bond isotope shifts. By virtue of the well-separated signals, the proton/deuteron fractional factors for both the SH and amide groups of the two Cys residues in EPPIb could be directly determined, as approximately 0.4-0.5 for [SD]/[SH] and 0.9-1.0 for [ND]/[NH], by the relative intensities of the NMR signals for the isotopomers. The proton NOE's of the two slowly exchanging SH's were clearly identified in the NOESY spectra and were useful for the determining the local structure of EPPIb around the Cys residues.

  8. On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS)*

    Science.gov (United States)

    Quanico, Jusal; Franck, Julien

    2016-01-01

    Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues. PMID:27512083

  9. On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS).

    Science.gov (United States)

    Quanico, Jusal; Franck, Julien; Salzet, Michel; Fournier, Isabelle

    2016-10-01

    Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid.

    Science.gov (United States)

    Wang, Lei; Murai, Yuta; Yoshida, Takuma; Okamoto, Masashi; Masuda, Katsuyoshi; Sakihama, Yasuko; Hashidoko, Yasuyuki; Hatanaka, Yasumaru; Hashimoto, Makoto

    2014-01-01

    In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives. N-Acetyl-protected natural aromatic α-amino acids (Tyr and Trp) were more effective in H/D exchange than unprotected ones. The N-acetylated TFMD phenylalanine derivative afforded slightly higher H/D exchange than unprotected derivatives. An effective post-deuteration method for cross-linkable α-amino acid derivatives will be useful for the analysis of biological functions of bioactive peptides and proteins by mass spectrometry.

  11. Hydrogen isotope exchange in tungsten: Discussion as removal method for tritium

    Science.gov (United States)

    Roth, J.; Schwarz-Selinger, T.; Alimov, V. Kh.; Markina, E.

    2013-01-01

    Hydrogen isotope exchange in re-crystallized polycrystalline tungsten was investigated at 320 and 450 K. In a first step the tungsten samples were loaded with deuterium to a fluence of 1024 D/m2 from a low-temperature plasma at 200 eV/D particle energy. In a second step, H was implanted at the same particle energy and similar target temperature with a mass-separated ion beam at different ion fluences ranging from 2 × 1020 to 7.5 × 1023 H/m2. The analytic methods used were nuclear reaction analysis with D(3He,p)α reaction and elastic recoil detection analysis with 4He. In order to determine the D concentration at depths of up to 7.4 μm the 3He energy was varied from 0.5 to 4.5 MeV. It was found that already at an H fluence of 2 × 1020 H/m2, i.e. at 1/5000 of the initial D fluence, about 30% of the retained D was released. Depth profiling of D without and with subsequent H implantation shows strong replacement close to the surface at 320 K, but extending to all analyzable depths at 450 K especially at high fluences, leading to higher release efficiency. The reverse sequence of hydrogen isotopes allowed the analysis of the replacing isotope and showed that the release of D is balanced by the uptake of H. It also shows that hydrogen does not diffuse through a region of filled traps into a region were unfilled traps can be encounter but transport is rather a dynamic process of trapping and de-trapping even at 320 K. Initial D retention in H loaded W is an order of magnitude higher than in pristine W, indicating that every H-containing trap is a potential trap for D. In consequence, hydrogen isotope exchange is not a viable method to significantly enhance the operation time before the tritium inventory limit is reached but should be considered an option to reduce the tritium inventory in ITER before major interventions at the end of an operation period.

  12. Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

    2016-01-01

    The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

  13. Hydrogen--deuterium exchange in saturated hydrocarbons on α-chromia catalyst

    International Nuclear Information System (INIS)

    Kalman, J.; Guczi, L.

    1977-01-01

    The kinetics of hydrogen--deuterium exchange in methane, ethane, and propane have been studied with unsupported α-chromia as catalyst in the temperature range of 598 to 688 0 K. The apparent activation energies for methane, ethane, and propane are 88, 130, and 84 kJ mol -1 , respectively, similar to those found on chromia gel. The order of reaction with respect to hydrocarbons and deuterium has been determined as also having the kinetic isotope effect. The main initial products are CH 3 D and CD 4 with methane, C 2 H 4 D 2 and C 2 D 6 with ethane, and C 3 H 7 D and C 3 D 8 with propane. A change in product distribution as a function of temperature, conversion, aging, and oxygen--deuterium treatment has been observed. In agreement with the kinetic data and the effect of oxygen, Cr 3+ has been assumed as the active species of the chromia catalyst, whereas Cr 2+ is an inactive site on the surface. There is no direct proof that chromium ion in a valence state higher than 3+ plays an important role in the exchange reaction. It was established that dual Cr 3+ -- Cr 3+ sites are responsible for the formation of ethane-d 2 . After a long deuterium treatment the number of dual sites is decreased, the effect being revealed in the decreased rate of exchange and the small amount of ethane-d 2 . On the contrary, the formation of methane-d 4 is interpreted by the formation of Cr = C bonds by analogy with homogeneous complexes. In the case of propane, the exchange reaction can be adequately interpreted by a π-allyl mechanism

  14. Gas-phase hydrogen/deuterium exchange of dinucleotides and 5'-monophosphate dinucleotides in a quadrupole ion trap

    Science.gov (United States)

    Chipuk, Joseph E.; Brodbelt, Jennifer S.

    2009-10-01

    Gas-phase hydrogen/deuterium (H/D) exchange reactions of four deprotonated dinucleotides (dAA, dAG, dGA, dGG) and their 5'-monophosphate analogs (5'-dAA, 5'-dAG, 5'-dGA, 5'-dGG) with D2O were performed in a quadrupole ion trap mass spectrometer. Significant differences in the rates and extents of exchange were found when the 5'-hydroxyl group of the dinucleotides was replaced by a phosphate functionality. Extensive and nucleobase-dependent exchange occurred for the deprotonated 5'-monophosphate dinucleotides, whereas the dinucleotides all exhibited essentially the same limited exchange. Results for the isomeric 5'-monophosphates, 5'-dAG and 5'-dGA, were remarkably different, indicating that the H/D exchange reaction was sequence dependent. An elaborate array of computations was performed to investigate the gas-phase structures of the ions individually and also as participants in ion-molecule complexes with D2O. Integration of the experimental and theoretical results supports a relay exchange mechanism and suggests that the exchange behavior depends highly on the identity and sequence of the nucleobases as well as their ability to interact with the deprotonation site. Finally, a shuttling mechanism is proposed to possibly account for the bimodal H/D exchange behavior observed for deprotonated 5'P-dGA. In this case, hydrogen bonding between the nucleobases in concert with interaction from the deuterating agent creates an ion-molecule complex in which hydrogen and deuterium atoms may be shuttled amongst the hydrogen-bonded participants.

  15. Solid-state NMR and hydrogen-deuterium exchange in a bilayer-solubilized peptide: structural and mechanistic implications.

    OpenAIRE

    Cotten, M; Fu, R; Cross, T A

    1999-01-01

    Hydrogen-deuterium exchange has been monitored by solid-state NMR to investigate the structure of gramicidin M in a lipid bilayer and to investigate the mechanisms for polypeptide insertion into a lipid bilayer. Through exchange it is possible to observe 15N-2H dipolar interactions in oriented samples that yield precise structural constraints. In separate experiments the pulse sequence SFAM was used to measure dipolar distances in this structure, showing that the dimer is antiparallel. The co...

  16. Proceedings of the 1st JAEA/KAERI information exchange meeting on HTGR and nuclear hydrogen technology

    International Nuclear Information System (INIS)

    Sato, Hiroyuki; Sakaba, Nariaki; Nishihara, Tetsuo; Yan, Xing L.; Hino, Ryutaro

    2007-03-01

    Japan Atomic Energy Agency (JAEA) has completed an implementation with Korea Atomic Energy Research Institute (KAERI) on HTGR and nuclear hydrogen technology, 'The Implementation of Cooperative Program in the Field of Peaceful Uses of Nuclear Energy between KAERI and JAEA. 'To facilitate efficient technology development on HTGR and nuclear hydrogen by the IS process, an information exchange meeting was held at the Oarai Research and Development Center of JAEA on August 28-30, 2006 under Program 13th of the JAEA/KAERI Implementation, 'Development of HTGR and Nuclear Hydrogen Technology'. JAEA and KAERI mutually showed the status and future plan of the HTTR (High-Temperature Engineering Test Reactor) project in Japan and of the NHDD (Nuclear Hydrogen Development and Demonstration) project in Korea, respectively, and discussed collaboration items. This proceedings summarizes all materials of presented technical discussions on HTGR and hydrogen production technology as well as the meeting briefing including collaboration items. (author)

  17. Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development - A review.

    Science.gov (United States)

    Deng, Bin; Lento, Cristina; Wilson, Derek J

    2016-10-12

    Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways. In manufacturing and development, HDX-MS can validate storage formulations and manufacturing processes for various biotherapeutics. Currently, HDX-MS is being refined to provide additional coverage, sensitivity and structural specificity and implemented on the millisecond timescale to reveal residual structure and dynamics in disordered domains and intrinsically disordered proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Structural changes of ultrasonicated bovine serum albumin revealed by hydrogen-deuterium exchange and mass spectrometry.

    Science.gov (United States)

    Zhang, Qiuting; Tu, Zongcai; Wang, Hui; Huang, Xiaoqin; Sha, Xiaomei; Xiao, Hui

    2014-11-01

    The structural changes of bovine serum albumin (BSA) under high-intensity ultrasonication were investigated by fluorescence spectroscopy and mass spectrometry. Evidence for the ultrasonication-induced conformational changes of BSA was provided by the intensity changes and maximum-wavelength shift in fluorescence spectrometry. Matrix-assisted laser desorption-ionization time-of-flight mass spectroscopy (MALDI-TOF MS) revealed the increased intensity of the peak at the charge state +5 and a newly emerged peak at charge state +6, indicating that the protein became unfolded after ultrasonication. Prevalent unfolding of BSA after ultrasonication was revealed by hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS). Increased intensity and duration of ultrasonication further promoted the unfolding of the protein. The unfolding induced by ultrasonication goes through an intermediate state similar to that induced by a low concentration of denaturant.

  19. Site-Specific Hydrogen Isotope Composition of Propane: Mass spectrometric methods, equilibrium temperature dependence, and kinetics of exchange

    Science.gov (United States)

    Xie, H.; Ponton, C.; Kitchen, N.; Lloyd, M. K.; Lawson, M.; Formolo, M. J.; Eiler, J. M.

    2016-12-01

    Intramolecular isotope ordering can constrain temperatures of synthesis, mechanisms of formation, and/or source substrates of organic compounds. Here we explore site-specific hydrogen isotope variations of propane. Statistical thermodynamic models predict that at equilibrium methylene hydrogen (-CH2-) in propane will be 10's of per mil higher in D/H ratio than methyl hydrogen (-CH3) at geologically relevant temperatures, and that this difference is highly temperature dependent ( 0.5-1 ‰/°C). Chemical-kinetic controls on site-specific D/H in propane could constrain the mechanisms, conditions and extents of propane synthesis or destruction. We have developed a method for measuring the difference in D/H ratio between methylene and methyl hydrogen in propane by gas source mass spectrometry. The data were measured using the Thermo Fisher Double Focusing Sector high resolution mass spectrometer (DFS), and involve comparison of the D/H ratios of molecular ion (C3H8+) and the ethyl fragmental ion (C2H5+). We demonstrate the accuracy and precision of this method through analysis of D-labeled and independently analyzed propanes. In the exchange experiments, propane was heated (100-200 oC) either alone or in the presence of D-enriched water (δD=1,1419 ‰ SMOW), with or without one of several potentially catalytic substrates for hours to weeks. Propane was found to exchange hydrogen with water vigorously at 200 °C in the presence of metal catalysts. In the presence of Ni catalyst, methylene hydrogen exchanges 2.5 times faster than methyl hydrogen. Hydrogen exchange in the presence of Pd catalyst is more effective and can equilibrate hydrogen isotope distribution on propane on the order of 7 days. Isotopic exchange in the presence of natural materials have also been tested, but is only measurable in the methylene group at 200 °C. High catalytic activity of Pd permits attainment of a bracketed, time-invariant equilibrium state that we use to calibrate the site

  20. Dissection of conformational conversion events during prion amyloid fibril formation using hydrogen exchange and mass spectrometry.

    Science.gov (United States)

    Singh, Jogender; Udgaonkar, Jayant B

    2013-09-23

    A molecular understanding of prion diseases requires an understanding of the mechanism of amyloid fibril formation by the prion protein. In particular, it is necessary to define the sequence of the structural events describing the conformational conversion of monomeric PrP to aggregated PrP. In this study, the sequence of the structural events in the case of amyloid fibril formation by recombinant mouse prion protein at pH7 has been characterized by hydrogen-deuterium exchange and mass spectrometry. The observation that fibrils are substantially more stable to hydrogen-deuterium exchange than is native monomer allows both forms to be quantified during the course of the aggregation reaction. Under the aggregation conditions utilized, native monomeric protein and amyloid fibrils are the only forms of the protein detectable during the course of the fibril formation reaction, suggesting that monomer directly adds on to the fibril template. Conformational conversion is shown to occur in two steps after the binding of monomer to fibril, with helix 1 unfolding only after helices 2 and 3 transform into β-sheet. Local stability in the β-sheet core region (residues ~159-225) of the fibrils is shown to be sequence dependent in that it varies along the length of the core, and local stability in protein molecules that are ordered in the structurally heterogeneous sequence segment 109-132 is shown to be similar to that in the core. This new understanding of the structural events during prion protein aggregation has important bearing on our comprehension of the molecular basis of prion pathogenesis. © 2013 Elsevier Ltd. All rights reserved.

  1. [Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

    Science.gov (United States)

    Zolotarev, Yu A; Dadayan, A K; Kozik, V S; Gasanov, E V; Nazimov, I V; Ziganshin, R Kh; Vaskovsky, B V; Murashov, A N; Ksenofontov, A L; Haribin, O N; Nikolaev, E N; Myasoedov, N F

    2014-01-01

    The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin β-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the β-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

  2. Comparison of experimental and theoretical data on hydrogen-deuterium exchange for ten globular proteins.

    Science.gov (United States)

    Suvorina, M Yu; Surin, A K; Dovidchenko, N V; Lobanov, M Yu; Galzitskaya, O V

    2012-06-01

    The number of protons available for hydrogen-deuterium exchange was predicted for ten globular proteins using a method described elsewhere by the authors. The average number of protons replaced by deuterium was also determined by mass spectrometry of the intact proteins in their native conformations. Based on these data, we find that two models proposed earlier agree with each other in estimation of the number of protons replaced by deuterium. Using a model with a probability scale for hydrogen bond formation, we estimated a number of protons replaced by deuterium that is close to the experimental data for long-term incubation in D(2)O (24 h). Using a model based on estimations with a scale of the expected number of contacts in globular proteins there is better agreement with the experimental data obtained for a short period of incubation in D(2)O (15 min). Therefore, the former model determines weakly fluctuating parts of a protein that are in contact with solvent only for a small fraction of the time. The latter model (based on the scale of expected number of contacts) predicts either flexible parts of a protein chain exposed to interactions with solvent or disordered parts of the protein.

  3. Deuterium-hydrogen exchange and scrambling reactions in the pyrolysis of labeled cyclopentene. A radical mechanism

    International Nuclear Information System (INIS)

    Kosnik, K.G.; Benson, S.W.

    1983-01-01

    Inter- and intraradical mechanisms that promote deuterium-hydrogen scrambling in labeled cyclopentene-d 1 are investigated at 800 and 1200 K. Rate constants are estimated for each step and for possible competing side reactions. The fast radical bimolecular exchange at 800 K and unimolecular scrambling at 1200 K of labeled cyclopentene are shown to be faster than the Woodward-Hoffman allowed 1,4 concerted molecular elimination of hydrogen. The low-energy estimate of 8.0 kcal/mol by Lewis of the difference between the allowed 1,4 and disallowed 1,2 channels can thus be explained. No conclusions can be drawn concerning the 1,2 channel. The estimated rates of radical reactions are in agreement with experiments on the addition of D 2 to cyclopentadiene at 300 0 C which shows only cis, 3 to 5 addition and with pyrolysis experiments at 500 +- 20 0 C which show about 5% contribution of a higher than first-order radical reaction. The mechanism of this radical decomposition is given and its steps are explicitly evaluated. 2 tables

  4. Gas-Phase Hydrogen/Deuterium Exchange of Dinucleotides and 5′-Monophosphate Dinucleotides in a Quadrupole Ion Trap

    OpenAIRE

    Chipuk, Joseph E.; Brodbelt, Jennifer S.

    2009-01-01

    Gas-phase hydrogen/deuterium (H/D) exchange reactions of four deprotonated dinucleotides (dAA, dAG, dGA, dGG) and their 5′-monophosphate analogs (5′-dAA, 5′-dAG, 5′-dGA, 5′-dGG) with D2O were performed in a quadrupole ion trap mass spectrometer. Significant differences in the rates and extents of exchange were found when the 5′-hydroxyl group of the dinucleotides was replaced by a phosphate functionality. Extensive and nucleobase-dependent exchange occurred for the deprotonated 5′-monophospha...

  5. Analysis of Overlapped and Noisy Hydrogen/Deuterium Exchange Mass Spectra

    Science.gov (United States)

    Guttman, Miklos; Weis, David D.; Engen, John R.; Lee, Kelly K.

    2013-12-01

    Noisy and overlapped mass spectrometry data hinder the sequence coverage that can be obtained from hydrogen deuterium exchange analysis, and places a limit on the complexity of the samples that can be studied by this technique. Advances in instrumentation have addressed these limits, but as the complexity of the biological samples under investigation increases, these problems are re-encountered. Here we describe the use of binomial distribution fitting with asymmetric linear squares regression for calculating the accurate deuterium content for mass envelopes of low signal or that contain significant overlap. The approach is demonstrated with a test data set of HIV Env gp140 wherein inclusion of the new analysis regime resulted in obtaining exchange data for 42 additional peptides, improving the sequence coverage by 11 %. At the same time, the precision of deuterium uptake measurements was improved for nearly every peptide examined. The improved processing algorithms also provide an efficient method for deconvolution of bimodal mass envelopes and EX1 kinetic signatures. All these functions and visualization tools have been implemented in the new version of the freely available software, HX-Express v2.

  6. Pulsed hydrogen/deuterium exchange mass spectrometry for time-resolved membrane protein folding studies.

    Science.gov (United States)

    Khanal, Anil; Pan, Yan; Brown, Leonid S; Konermann, Lars

    2012-12-01

    Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D(2) O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N-H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry.

    Science.gov (United States)

    Bou-Assaf, George M; Chamoun, Jean E; Emmett, Mark R; Fajer, Piotr G; Marshall, Alan G

    2010-04-15

    Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein-protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of (13)C and/or (15)N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ((12)C(c)(1)H(h)(14)N(n)(16)O(o)(32)S(s)), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.

  8. Analysis of overlapped and noisy hydrogen/deuterium exchange mass spectra.

    Science.gov (United States)

    Guttman, Miklos; Weis, David D; Engen, John R; Lee, Kelly K

    2013-12-01

    Noisy and overlapped mass spectrometry data hinder the sequence coverage that can be obtained from hydrogen deuterium exchange analysis, and places a limit on the complexity of the samples that can be studied by this technique. Advances in instrumentation have addressed these limits, but as the complexity of the biological samples under investigation increases, these problems are re-encountered. Here we describe the use of binomial distribution fitting with asymmetric linear squares regression for calculating the accurate deuterium content for mass envelopes of low signal or that contain significant overlap. The approach is demonstrated with a test data set of HIV Env gp140 wherein inclusion of the new analysis regime resulted in obtaining exchange data for 42 additional peptides, improving the sequence coverage by 11%. At the same time, the precision of deuterium uptake measurements was improved for nearly every peptide examined. The improved processing algorithms also provide an efficient method for deconvolution of bimodal mass envelopes and EX1 kinetic signatures. All these functions and visualization tools have been implemented in the new version of the freely available software, HX-Express v2.

  9. The deuterium-exchange reaction between water and hydrogen with the thin-film hydrophobic catalyst

    International Nuclear Information System (INIS)

    Yamashita, Hisao; Mizumoto, Mamoru; Matsuda, Shimpei

    1985-01-01

    The deuterium-exchange reaction between water and hydrogen with a hydrophobic catalyst was studied. The hydrophobic catalyst was composed of platinum as an active component and porous poly(tetrafluoroethylene) (PTFE) as a support. The PTFE support was in two forms, i.e., (a) a pellet and (b) a thin-film with the thickness of 50 μm. The primary purpose of the thin film hydrophobic catalyst was to reduce the platinum usage in the reactor. The activity of the catalyst was measured in a trickle bed reactor at atmospheric pressure and temperature of 20 ∼ 70 deg C. It has been found that the employment of the thin-film catalyst reduced the platinum usage to 1/5 of the reactor in the case of using a conventional catalyst. Platinum particles on the thin-film catalyst work efficiently because the reactants were easily diffused to the active sites. It has also been found that the isotopic exchange rate with the thin-film catalyst increased with the increase in the ratio of liquid/gas and increased with the rise of the reaction temperature. It was found from an endurance test that the activity of the thin-film catalyst decreased gradually due to the condensation of water vapor in the catalyst, but was regenarated by heating the catalyst to remove the condensed water. (author)

  10. Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry—A review

    International Nuclear Information System (INIS)

    Percy, Andrew J.; Rey, Martial; Burns, Kyle M.; Schriemer, David C.

    2012-01-01

    Highlights: ► Protein chemistry generates mass shifts useful for structure–function studies. ► H/DX supports a powerful mass shift method for protein interaction analysis. ► H/DX mass shifts are useful for determining binding data (K d , off-rates). ► Improved H/DX–MS workflows can accommodate complex protein systems. - Abstract: Assessing the functional outcome of protein interactions in structural terms is a goal of structural biology, however most techniques have a limited capacity for making structure–function determinations with both high resolution and high throughput. Mass spectrometry can be applied as a reader of protein chemistries in order to fill this void, and enable methodologies whereby protein structure–function determinations may be made on a proteome-wide level. Protein hydrogen/deuterium exchange (H/DX) offers a chemical labeling strategy suitable for tracking changes in “dynamic topography” and thus represents a powerful means of monitoring protein structure–function relationships. This review presents the exchange method in the context of interaction analysis. Applications involving interface detection, quantitation of binding, and conformational responses to ligation are discussed, and commentary on recent analytical developments is provided.

  11. Ammonia-water exchange front end process for ammonia-hydrogen heavy water plants (Preprint No. PD-1)

    International Nuclear Information System (INIS)

    Sadhukhan, H.K.; Varadarajan, T.G.; Nair, N.K.; Das, S.K.; Nath, G.K.

    1989-04-01

    The ammonia-hydrogen exchange process, which utilizes the deutrium exchange between liquid ammonia and gaseous hydrogen is a parasitic process and the heavy water plants (HWP) based on this process has to be linked with the fertilizer plant (FP) for its enormous requirements of hydrogen (synthesis gas, N 2 +3H 2 ). This dependence of HWP on FP gives rise to certain constraints which are listed. These deficiencies of the ammonia-hydrogen process can be overcome to a great extent by delinking the HWP from FP by incorporating NH 3 -H 2 O exchange as the front end step. In addition to the elimination of the above limitations, by employing water as the ultimate feed for the HWP, the plant capacity can be increased substantially and this would go a long way in achieving economies of the large capacity plants. A schematic diagram of this integrated plant is given. Some of the results of developmental efforts and feasibility studies of this NH 3 -H 2 O exchange are briefly reviewed. (author). 4 figs

  12. Ruthenium-catalyzed hydrogen isotope exchange of C(sp3)-H bonds directed by a sulfur atom.

    Science.gov (United States)

    Gao, Longhui; Perato, Serge; Garcia-Argote, Sébastien; Taglang, Céline; Martínez-Prieto, Luis Miguel; Chollet, Céline; Buisson, David-Alexandre; Dauvois, Vincent; Lesot, Philippe; Chaudret, Bruno; Rousseau, Bernard; Feuillastre, Sophie; Pieters, Grégory

    2018-03-25

    We present here the first example of C(sp 3 )-H activation directed by a sulfur atom. Based on this transformation catalyzed by Ru/C, we have developed a hydrogen isotope exchange reaction for the deuterium and tritium labelling of thioether substructures in complex molecules.

  13. Pinpointing changes in higher-order protein structure by hydrogen/deuterium exchange coupled to electron transfer dissociation mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper Dyrberg

    2013-01-01

    This Feature describes the use of electron transfer dissociation (ETD) to analyze the hydrogen/deuterium exchange (HDX) of proteins at increased spatial resolution down to the level of individual residues. A practical overview of how to couple ETD to the classical bottom-up HDX-MS workflow is given...

  14. Gaseous anion chemistry. Hydrogen-deuterium exchange in mono- and dialcohol alkoxide ions: ionization reactions in dialcohols

    International Nuclear Information System (INIS)

    Lloyd, J.R.; Agosta, W.C.; Field, F.H.

    1980-01-01

    The subject of this work is H-D exchange in certain gaseous anions using D 2 as the exchanging agent. The anions involved are produced from ethylene glycol, 1,3-propanediol, 1,4-butanediol, ethanol, 1-propanol, and 1-butanol. Spectra and postulated ionization reactions for these mono- and dialcohols are given. Hydrogen-deuterium exchange occurs in the (M - 1) - and (2M - 1) - ions of ethylene glycol, 1,3-propanediol, and 1,4-butanediol. The amount of exchange occurring is 3-8 times greater in (2M - 1) - than in (M - 1) - . The amount of H-D exchange occurring in ethanol, 1-propanol, and 1-butanol is small or zero in the (2M - 1) - ions and in the (M - 1) - ion for 1-butanol [the only (M - 1) - ion which could be examined experimentally]. The amount of exchange occurring in the (2M - 1) - and (M - 1) - ions from ethylene glycol is not affected by the total pressure or composition of the reaction mixture in the ionization chamber of the mass spectrometer. A novel hydrogen-bridging mechanism is suggested to account for the observed exchange occurring in the dialcohols

  15. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman

    2015-01-01

    in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation......-type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution....... to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found...

  16. Water-Protein Hydrogen Exchange in the Micro-Crystalline Protein Crh as Observed by Solid State NMR Spectroscopy

    International Nuclear Information System (INIS)

    Boeckmann, Anja; Juy, Michel; Bettler, Emmanuel; Emsley, Lyndon; Galinier, Anne; Penin, Francois; Lesage, Anne

    2005-01-01

    We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T 2 ' -selective 1 H- 13 C- 13 C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T 2 ' selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on 13 C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates

  17. Destruction of gel sulfonated cation-exchangers of the KU-2 type under the influence of hydrogen peroxide

    Energy Technology Data Exchange (ETDEWEB)

    Roginskaya, B.S.; Zavadovskaya, A.S.; Znamenskii, Yu.P.; Paskhina, N.A.; Dobrova, E.I.

    1988-10-20

    The purpose of this work was to study the mechanism of interaction of Soviet sulfonated cation-exchangers of the KU-2 type with hydrogen peroxide. It is shown that under the influence of hydrogen peroxide sulfonated cation-exchangers begin, after a certain induction period, to lose capacity and to release destruction products into water; the length of the induction period increases with the degree of cross-linking. In a given time of contact between the resin and the solution the degree of destruction falls with increase of cross-linking. The principal product of destruction of sulfonated cation-exchangers is an aromatic sulfonic acid containing oxidized groups in the side chains.

  18. NMR measurements of proton exchange between solvent and peptides and proteins

    International Nuclear Information System (INIS)

    Wojcik, J.; Ruszczynska, K.; Zhukov, I.; Ejchart, A.

    1999-01-01

    Scope and limitations of the NMR based methods, equilibration and magnetization transfer, for measuring proton exchange rates of amide protons in peptides and proteins with water protons are discussed. Equilibration is applied to very slow processes detected by hydrogen-deuterium exchange after a solute is dissolved in D 2 O. Magnetization transfer allows to study moderately rapid processes in H 2 O. A number of precautions should be undertaken in order to avoid systemic errors inherent in the magnetization transfer method. (author)

  19. Towards quantifying the role of exact exchange in the prediction hydrogen bond spin-spin coupling constants involving fluorine

    Energy Technology Data Exchange (ETDEWEB)

    San Fabián, J.; Omar, S.; García de la Vega, J. M., E-mail: garcia.delavega@uam.es [Departamento de Química Física Aplicada, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid (Spain)

    2016-08-28

    The effect of a fraction of Hartree-Fock exchange on the calculated spin-spin coupling constants involving fluorine through a hydrogen bond is analyzed in detail. Coupling constants calculated using wavefunction methods are revisited in order to get high-level calculations using the same basis set. Accurate MCSCF results are obtained using an additive approach. These constants and their contributions are used as a reference for density functional calculations. Within the density functional theory, the Hartree-Fock exchange functional is split in short- and long-range using a modified version of the Coulomb-attenuating method with the SLYP functional as well as with the original B3LYP. Results support the difficulties for calculating hydrogen bond coupling constants using density functional methods when fluorine nuclei are involved. Coupling constants are very sensitive to the Hartree-Fock exchange and it seems that, contrary to other properties, it is important to include this exchange for short-range interactions. Best functionals are tested in two different groups of complexes: those related with anionic clusters of type [F(HF){sub n}]{sup −} and those formed by difluoroacetylene and either one or two hydrogen fluoride molecules.

  20. Direct-hydrogen-fueled proton-exchange-membrane fuel cell system for transportation applications. Hydrogen vehicle safety report

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, C.E. [Directed Technologies, Inc., Arlington, VA (United States)

    1997-05-01

    This report reviews the safety characteristics of hydrogen as an energy carrier for a fuel cell vehicle (FCV), with emphasis on high pressure gaseous hydrogen onboard storage. The authors consider normal operation of the vehicle in addition to refueling, collisions, operation in tunnels, and storage in garages. They identify the most likely risks and failure modes leading to hazardous conditions, and provide potential countermeasures in the vehicle design to prevent or substantially reduce the consequences of each plausible failure mode. They then compare the risks of hydrogen with those of more common motor vehicle fuels including gasoline, propane, and natural gas.

  1. Study of hydrogen mobility by hydrogen-deuterium exchange. II. Theoretical kinetic study in alkyl and amino-alkyl pyrimidines

    International Nuclear Information System (INIS)

    Pompon, Alain

    1975-01-01

    Alkyl groups bound to the pyrimidine ring can be deuterium substituted on the carbon adjacent to the ring, in acidic D 2 O; kinetic equations corresponding to various exchange mechanism hypothesis are established. It is shown that theoretical and experimental results can be compared in order to precise the mechanism and to measure the characteristic parameters of the exchange reaction [fr

  2. Orientation in metal-catalyzed hydrogen exchange between alkanes, naphthalene, or biphenyl and deuterium or deuterium oxide

    International Nuclear Information System (INIS)

    Long, M.A.; Moyes, R.B.; Wells, P.B.; Garnett, J.L.

    1978-01-01

    Hydrogen isotope exchange between deuterium gas and protium in hexane, pentane, 2-methyl-butane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane, and 2,4-dimethylpentane has been catalyzed by clean platinum films (70--100 0 C). A selection of these reactions has been catalyzed by films of rhodium and iridium (typically -13 to -35 0 C). In all cases, multiple exchange occurred. Product analysis by mass and proton NMR spectroscopy showed that exchange in methylene and methine groups was more rapid than that in methyl groups. A similar orientation effect was observed in reactions over platinum powder but not over platinum-alumina. For exchange between deuterium oxide and hexane catalyzed by platinum films at 200 0 C, the rate of exchange in methyl groups exceeded that in methylene groups. It is proposed that preferential exchange in methylene and methine groups is normal behavior during alkane exchange with molecular deuterium over these platinum metals when their surfaces (i) are initially clean or (ii) contain several adjacent sites which are unperturbed by the presence of any electronegative adsorbed species. Preferential exchange in the methyl groups of hexane results from contamination of the surface by adsorbed D 2 O, OD, or O; this may be a geometric effect or an electronic effect, depending on the magnitude of the surface coverage of water, which is unknown. Orientation in the exchange between deuterium gas and naphthalene or biphenyl catalyzed by films of platinum and iridium does not differ from that observed in exchanges where the isotope source is deuterium oxide or deuterated solvent, but the M value calculated for exchange in naphthalene is higher than that previously reported

  3. Applications of the water--gas shift reaction. II. Catalytic exchange of deuterium for hydrogen at saturated carbon

    International Nuclear Information System (INIS)

    Laine, R.M.; Thomas, D.W.; Cary, L.W.; Buttrill, S.E.

    1978-01-01

    Previous studies on the homogeneous catalysis of the water-gas shift reaction by metal complexes of groups 6 and 8 had been carried out using aqueous alcoholic solutions of group 8 metal carbonyl complexes made basic with KOH. Substitution of triethylamine (Et 3 N) for KOH as base and alcohol for solvent led to the discovery that Et 3 N in the presence of D 2 O, CO, and Rh 6 (CO) 16 at 150 0 C undergoes an unusual catalytic exchange of deuterium for hydrogen. A suggested mechanism for this reaction is given and includes activation of hydrogen at a saturated carbon

  4. Hydrogen-deuterium exchange reaction of 2-methylpyridine catalyzed by several fatty acids

    International Nuclear Information System (INIS)

    Hirata, Hirohumi; Fukuzumi, Kazuo.

    1976-01-01

    Hydrogen-deuterium exchange reaction of 2-methylpyridine has been studied by using several fatty acids as catalysts. The reaction was carried out in a sealed pyrex tube at 120 0 C, and the contents of the products were determined by mass spectrometry. Reaction of 2-methylpyridine with monodeuteroacetic acid (1 : 1, mol/mol) arrived at a equilibrium (d 0 reversible d 1 reversible d 2 reversible d 3 ) in 2 hr (d 0 41%, d 1 42%, d 2 15%, d 3 2%). No exchange was observed for the reaction of pyridine with monodeuteroacetic acid. The conversion-time curves of typical series reactions (d 0 → d 1 → d 2 → d 3 ) were obtained for the fatty acid catalyzed exchange in deuterium oxide. The effect of the fatty acid RCO 2 H (substrate : fatty acid : D 2 O=1 : 0.86 : 27.6, mol/mol/mol) on the conversion was in the order of R; C 1 --C 3 4 --C 10 , where the reaction mixtures were homogeneous in the case of C 1 --C 3 and were heterogeneous in the case of C 4 --C 10 . The effects of the initial concentration of the substrates and the catalysts (RCO 2 H) on the total conversion were studied by using some fatty acids (R; C 2 , C 4 and C 9 ) in deuterium oxide (for 2 hr). The total conversion of the substrate increases with increasing the concentration of the acids. The total conversion decreases in the case of R=C 9 , but, increases in the case of R=C 2 with increasing the concentration of the substrate. In the case of reactions with low concentrations of the substrate, the reactivity was in the order of C 9 >C 4 >C 2 , while with high concentrations, the reactivity was in the order of C 4 >C 2 >C 9 and C 9 >C 4 >C 2 with high and low concentrations of the acids, respectively. A possible reaction mechanism was proposed and discussed. (auth.)

  5. Controlling hydrogen scrambling in multiply charged protein ions during collisional activation: implications for top-down hydrogen/deuterium exchange MS utilizing collisional activation in the gas phase.

    Science.gov (United States)

    Abzalimov, Rinat R; Kaltashov, Igor A

    2010-02-01

    Hydrogen exchange in solution combined with ion fragmentation in the gas phase followed by MS detection emerged in recent years as a powerful tool to study higher order protein structure and dynamics. However, a certain type of ion chemistry in the gas phase, namely, internal rearrangement of labile hydrogen atoms (the so-called hydrogen scrambling), is often cited as a factor limiting the utility of this experimental technique. Although several studies have been carried out to elucidate the roles played by various factors in the occurrence and the extent of hydrogen scrambling, there is still no consensus as to what experimental protocol should be followed to avoid or minimize it. In this study we employ fragmentation of mass-selected subpopulations of protein ions to assess the extent of internal proton mobility prior to dissociation. A unique advantage of tandem MS is that it not only provides a means to map the deuterium content of protein ions whose overall levels of isotope incorporation can be precisely defined by controlling the mass selection window, but also correlates this spatial isotope distribution with such global characteristic as the protein ion charge state. Hydrogen scrambling does not occur when the charge state of the precursor protein ions selected for fragmentation is high. Fragment ions derived from both N- and C-terminal parts of the protein are equally unaffected by scrambling. However, spatial distribution of deuterium atoms obtained by fragmenting low-charge-density protein ions is consistent with a very high degree of scrambling prior to the dissociation events. The extent of hydrogen scrambling is also high when multistage fragmentation is used to probe deuterium incorporation locally. Taken together, the experimental results provide a coherent picture of intramolecular processes occurring prior to the dissociation event and provide guidance for the design of experiments whose outcome is unaffected by hydrogen scrambling.

  6. Towards rotationally state-resolved differential cross sections for the hydrogen exchange reaction

    Energy Technology Data Exchange (ETDEWEB)

    Vrakking, Marcus Johannes Jacobus [Univ. of California, Berkeley, CA (United States)

    1992-11-01

    The hydrogen exchange reaction H + H2 → H2 + H (and its isotopic variants) plays a pivotal role in chemical reaction dynamics. It is the only chemical reaction for which fully converged quantum scattering calculations have been carried out using a potential energy surface which is considered to be chemically accurate. To improve our ability to test the theory, a `perfect experiment`, measuring differential cross sections with complete specification of the reactant and product states, is called for. In this thesis, the design of an experiment is described that aims at achieving this goal for the D + H2 reaction. A crossed molecular beam arrangement is used, in which a photolytic D atom beam is crossed by a pulsed beam of H2 molecules. DH molecules formed in the D + H2 reaction are state-specifically ionized using Doppler-free (2+1) Resonance-Enhanced Multi-Photon Ionization (REMPI) and detected using a Position-sensitive microchannel plate detector. This detection technique has an unprecedented single shot detection sensitivity of 6.8 103 molecules/cc. This thesis does not contain experimental results for the D + H2 reaction yet, but progress that has been made towards achieving this goal is reported. In addition, results are reported for a study of the Rydberg spectroscopy of the water molecule.

  7. Towards rotationally state-resolved differential cross sections for the hydrogen exchange reaction

    Energy Technology Data Exchange (ETDEWEB)

    Vrakking, M.J.J.

    1992-11-01

    The hydrogen exchange reaction H + H[sub 2] [yields] H[sub 2] + H (and its isotopic variants) plays a pivotal role in chemical reaction dynamics. It is the only chemical reaction for which fully converged quantum scattering calculations have been carried out using a potential energy surface which is considered to be chemically accurate. To improve our ability to test the theory, a 'perfect experiment', measuring differential cross sections with complete specification of the reactant and product states, is called for. In this thesis, the design of an experiment is described that aims at achieving this goal for the D + H[sub 2] reaction. A crossed molecular beam arrangement is used, in which a photolytic D atom beam is crossed by a pulsed beam of H[sub 2] molecules. DH molecules formed in the D + H[sub 2] reaction are state-specifically ionized using Doppler-free (2+1) Resonance-Enhanced Multi-Photon Ionization (REMPI) and detected using a Position-sensitive microchannel plate detector. This detection technique has an unprecedented single shot detection sensitivity of 6.8 10[sup 3] molecules/cc. This thesis does not contain experimental results for the D + H[sub 2] reaction yet, but progress that has been made towards achieving this goal is reported. In addition, results are reported for a study of the Rydberg spectroscopy of the water molecule.

  8. Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Huang, Richard Y.-C.; Iacob, Roxana E.; Sankaranarayanan, Sethu; Yang, Ling; Ahlijanian, Michael; Tao, Li; Tymiak, Adrienne A.; Chen, Guodong

    2018-01-01

    Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. [Figure not available: see fulltext.

  9. Conformations of JNK3α splice variants analyzed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Park, Ji Young; Yun, Youngjoo; Chung, Ka Young

    2017-03-01

    c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family that regulate apoptosis, inflammation, cytokine production, and metabolism. MAPKs undergo various splicing within their kinase domains. Unlike other MAPKs, JNKs have alternative splicing at the C-terminus, resulting in long and short variants. Functional or conformational effects due to the elongated C-terminal tail in the long splice variants have not been investigated nor has the conformation of the C-terminal tail been analyzed. Here, we analyzed the conformation of the elongated C-terminal tail and investigated conformational differences between long and short splice variants of JNKs using JNK3α2 and JNK3α1 as models. We adopted hydrogen/deuterium exchange mass spectrometry (HDX-MS) to analyze the conformation. HDX-MS revealed that the C-terminal tail is mostly intrinsically disordered, and that the conformation of the kinase domain of JNK3α2 is more dynamic than that of JNK3α1. The different conformation dynamics between long and short splice variants of JNK3α might affect the cellular functions of JNK3. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Online deuterium hydrogen exchange and protein digestion coupled with ion mobility spectrometry and tandem mass spectrometry.

    Science.gov (United States)

    Donohoe, Gregory C; Arndt, James R; Valentine, Stephen J

    2015-05-19

    Online deuterium hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within ∼60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment. Extracted ion drift time distributions (XIDTDs) of deuterated peptic peptides are mobility-matched to corresponding XIDTDs of undeuterated peptic peptides that were identified using collision-induced dissociation (CID). Matching XIDTDs allows a straightforward identification and deuterium retention evaluation for labeled peptides. Aside from the mobility separation, the ion trapping capabilities of the LTQ, combined with ETD, are demonstrated to provide single-residue resolution. Deuterium retention for the c- series ions across residues M(1)-L(15) and N(25)-R(42) are in good agreement with the known secondary structural elements within ubiquitin.

  11. Studies of negative ions by collision-induced decomposition and hydrogen-deuterium exchange techniques.

    Science.gov (United States)

    Hunt, D F; Sethi, S K; Shabanowitz, J

    1980-06-01

    Development of two new techniques for studying the gas phase chemistry of negative ions is reported. Collision induced dissociation (CID) of (M-1)- ions has been accomplished in a newly constructed triple stage quadrupole mass spectrometer. This instrument was assembled by adding two additional Finnigan quadrupole mass filters to a Finnigan Model 3200 CI mass spectrometer. Generation of (M-1)- ions is accomplished by allowing OH- and sample to react under CI conditions in the ion source. The first quadrupole mass filter, Q1, is then employed to selectively pass the (M-1)- ion into a second quadrupole filter containing argon or neon at 10(-3) torr. On collision with the inert gas the (M-1)- ions dissociate into fragments which are then mass analyzed in the third quadrupole filter, CID spectra of (M-1)- ions from twelve carbonyl compounds are presented in this paper. Ion molecule isotope exchange reactions in the CI ion source can be used to count the number of hydrogen atoms in many different chemical environments. Collisions between sample (M-1)- ions and deuterium-labeled reagent gases (ND3, D2O, EtOD) facilitate incorporation of deuterium into the negative ion if the basicities of the sample and reagent anions are similar. Thus it is possible to selectively incorporate deuterium into many organic samples by controlling the exothermicity of the acid base, ion-molecule chemistry.

  12. Differential hydrogen/deuterium exchange mass spectrometry analysis of protein-ligand interactions.

    Science.gov (United States)

    Chalmers, Michael J; Busby, Scott A; Pascal, Bruce D; West, Graham M; Griffin, Patrick R

    2011-02-01

    Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.

  13. Hydrogen-deuterium exchange reveals long-range dynamical allostery in soybean lipoxygenase.

    Science.gov (United States)

    Offenbacher, Adam R; Iavarone, Anthony T; Klinman, Judith P

    2018-01-26

    In lipoxygenases, the topologically conserved C-terminal domain catalyzes the oxidation of polyunsaturated fatty acids, generating an assortment of biologically relevant signaling mediators. Plant and animal lipoxygenases also contain a 100-150-amino acid N-terminal C2-like domain that has been implicated in interactions with isolated fatty acids and at the phospholipid bilayer. These interactions may lead to increased substrate availability and contribute to the regulation of active-site catalysis. Because of a lack of structural information, a molecular understanding of this lipid-protein interaction remains unresolved. Herein, we employed hydrogen-deuterium exchange MS (HDXMS) to spatially resolve changes in protein conformation upon interaction of soybean lipoxygenase with a fatty acid surrogate, oleyl sulfate (OS), previously shown to act at a site separate from the substrate-binding site. Specific, OS-induced conformational changes are detected both at the N-terminal domain and within the substrate portal nearly 30 Å away. Combining previously measured kinetic properties in the presence of OS with its impact on the K d for linoleic acid substrate binding, we conclude that OS binding brings about an increase in rate constants for both the ingress and egress of substrate. We discuss the role of OS-induced changes in protein flexibility in the context of changes in the mechanism of substrate acquisition. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. The structural basis of serpin polymerization studied by hydrogen/deuterium exchange and mass spectrometry.

    Science.gov (United States)

    Tsutsui, Yuko; Kuri, Barbara; Sengupta, Tanusree; Wintrode, Patrick L

    2008-11-07

    The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type alpha(1)-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the alpha(1)-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of beta-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into beta-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of beta-sheet C. On the basis of our results, we propose a mechanism for polymerization in which beta-strand 1C is displaced from the rest of beta-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of beta-sheet A, and allows for subsequent polymerization.

  15. Functional unfolding of alpha1-antitrypsin probed by hydrogen-deuterium exchange coupled with mass spectrometry.

    Science.gov (United States)

    Baek, Je-Hyun; Yang, Won Suk; Lee, Cheolju; Yu, Myeong-Hee

    2009-05-01

    The native state of alpha(1)-antitrypsin (alpha(1)AT), a member of the serine protease inhibitor (serpin) family, is considered a kinetically trapped folding intermediate that converts to a more stable form upon complex formation with a target protease. Although previous structural and mutational studies of alpha(1)AT revealed the structural basis of the native strain and the kinetic trap, the mechanism of how the native molecule overcomes the kinetic barrier to reach the final stable conformation during complex formation remains unknown. We hypothesized that during complex formation, a substantial portion of the molecule undergoes unfolding, which we dubbed functional unfolding. Hydrogen-deuterium exchange coupled with ESI-MS was used to analyze this serpin in three forms: native, complexing, and complexed with bovine beta-trypsin. Comparing the deuterium content at the corresponding regions of these three samples, we probed the unfolding of alpha(1)AT during complex formation. A substantial portion of the alpha(1)AT molecule unfolded transiently during complex formation, including not only the regions expected from previous structural studies, such as the reactive site loop, helix F, and the following loop, but also regions not predicted previously, such as helix A, strand 6 of beta-sheet B, and the N terminus. Such unfolding of the native interactions may elevate the free energy level of the kinetically trapped native serpin sufficiently to cross the transition state during complex formation. In the current study, we provide evidence that protein unfolding has to accompany functional execution of the protein molecule.

  16. Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions

    Science.gov (United States)

    Chalmers, Michael J; Busby, Scott A; Pascal, Bruce D; West, Graham M; Griffin, Patrick R

    2011-01-01

    Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule–receptor interactions, this technique has also been applied to study protein–protein complexes, such as mapping antibody–antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein–ligand interactions has had an impact on biology and drug discovery. PMID:21329427

  17. Accurate Quantitation of Water-amide Proton Exchange Rates Using the Phase-Modulated CLEAN Chemical EXchange (CLEANEX-PM) Approach with a Fast-HSQC (FHSQC) Detection Scheme

    International Nuclear Information System (INIS)

    Hwang, Tsang-Lin; Zijl, Peter C.M. van; Mori, Susumu

    1998-01-01

    Measurement of exchange rates between water and NH protons by magnetization transfer methods is often complicated by artifacts, such as intramolecular NOEs, and/or TOCSY transfer from Cα protons coincident with the water frequency, or exchange-relayed NOEs from fast exchanging hydroxyl or amine protons. By applying the Phase-Modulated CLEAN chemical EXchange (CLEANEX-PM) spin-locking sequence, 135 o (x) 120 o (-x) 110 o (x) 110 o (-x) 120 o (x) 135 o (-x) during the mixing period, these artifacts can be eliminated, revealing an unambiguous water-NH exchange spectrum. In this paper, the CLEANEX-PM mixing scheme is combined with Fast-HSQC (FHSQC) detection and used to obtain accurate chemical exchange rates from the initial slope analysis for a sample of 15N labeled staphylococcal nuclease. The results are compared to rates obtained using Water EXchange filter (WEX) II-FHSQC, and spin-echo-filtered WEX II-FHSQC measurements, and clearly identify the spurious NOE contributions in the exchange system

  18. Quenched hydrogen-deuterium exchange NMR of a disease-relevant Aβ(1-42) amyloid polymorph.

    Science.gov (United States)

    Wälti, Marielle Aulikki; Orts, Julien; Riek, Roland

    2017-01-01

    Alzheimer's disease is associated with the aggregation into amyloid fibrils of Aβ(1-42) and Aβ(1-40) peptides. Interestingly, these fibrils often do not obtain one single structure but rather show different morphologies, so-called polymorphs. Here, we compare quenched hydrogen-deuterium (H/D) exchange of a disease-relevant Aβ(1-42) fibril for which the 3D structure has been determined by solid-state NMR with H/D exchange previously determined on another structural polymorph. This comparison reveals secondary structural differences between the two polymorphs suggesting that the two polymorphisms can be classified as segmental polymorphs.

  19. The exchange reaction between hydrogen and deuterium. I. Importance of surface reactions in the steady-state mechanism

    International Nuclear Information System (INIS)

    Marteau, Chantal; Gaillard-Cusin, Francoise; James, H.

    1978-01-01

    Investigation of heterogeneous initiation process of gas phase linear chain reactions is carried out through the study of H 2 -D 2 exchange reaction. Experimental data under study concern mainly the stationary rate of HD formation and the prestationary proceeding. Steady-state method accounts for the first one of these data; it allows to clearly compare the wall process part to the part played by the homogeneous chain reaction towards HD formation. Activation energy of exchange elementary step between chemisorbed hydrogen (on silica) and gaseous deuterium has been evaluated: Esub(e1)=52+-1 Kcal/mole [fr

  20. Process and device for stage by stage enrichment of deuterium and/or tritium in a material suitable for isotope exchange of deuterium and tritium with hydrogen

    International Nuclear Information System (INIS)

    Iniotakis, N.; Decken, C.B. von der.

    1983-01-01

    Water containing deuterium and/or tritium is first introduced into a carrier gas flow and reduced for the stage by stage enrichment of deuterium and/or tritium. A hydrogen partial pressure of a maximum of 100 millibar is set in the carrier gas flow. The carrier gas flow is taken along the primary side of an exchange wall suitable for the permeation of hydrogen, and a further carrier gas flow flows on its secondary side, which contains water or hydrogen. Reaction products formed after isotope exchange of deuterium and/or tritium with hydrogen are removed by the secondary carrier gas flow. (orig./HP) [de

  1. Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Masson, Glenn R; Maslen, Sarah L; Williams, Roger L

    2017-05-16

    Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics. © 2017 The Author(s).

  2. Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

    OpenAIRE

    Dautant , Alain; Meyer , Philippe; Georgescauld , Florian

    2017-01-01

    International audience; Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the WT and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding/folding by Hydrogen/Deuterium exchange mass spectrometry (HDX-MS...

  3. Dynamics and ligand-induced conformational changes in human prolyl oligopeptidase analyzed by hydrogen/deuterium exchange mass spectrometry

    OpenAIRE

    Tsirigotaki, Alexandra; Elzen, van, Roos; Veken, van der, Pieter; Lambeir, Anne-Marie; Economou, Anastassios

    2017-01-01

    Abstract: Prolyl oligopeptidase (PREP) is conserved in many organisms across life. It is involved in numerous processes including brain function and neuropathology, that require more than its strict proteolytic role. It consists of a seven-bladed beta-propeller juxtaposed to a catalytic alpha/beta-hydrolase domain. The conformational dynamics of PREP involved in domain motions and the gating mechanism that allows substrate accessibility remain elusive. Here we used Hydrogen Deuterium eXchange...

  4. Site-specific analysis of gas-phase hydrogen/deuterium exchange of peptides and proteins by electron transfer dissociation.

    Science.gov (United States)

    Rand, Kasper D; Pringle, Steven D; Morris, Michael; Brown, Jeffery M

    2012-02-21

    To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND(3) gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1-10 ms) in ND(3) gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both near-native and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research.

  5. Backbone amide linker strategy

    DEFF Research Database (Denmark)

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen

    2013-01-01

    In the backbone amide linker (BAL) strategy, the peptide is anchored not at the C-terminus but through a backbone amide, which leaves the C-terminal available for various modifications. This is thus a very general strategy for the introduction of C-terminal modifications. The BAL strategy...... to assemble the final peptide. One useful application of this strategy is in the synthesis of C-terminal peptide aldehydes. The C-terminal aldehyde is masked as an acetal during synthesis and then conveniently demasked in the final cleavage step to generate the free aldehyde. Another application...

  6. A study on the deactivation and stability of hydrophobic catalyst for hydrogen isotope exchange

    International Nuclear Information System (INIS)

    Sohn, Soon Hwan

    2006-02-01

    The hydrophobic catalyst has been prepared by deposition of platinum on porous styrene divinylbenzene copolymers(Pt/SDBC) and at the same time a separated type catalytic reactor has been developed for the Wolsong tritium removal facility(WTRF). Several tests carried out to obtain the experimental performance data of the Pt/SDBC with a recycle reactor system. The long-term stability was also measured with the Pt/SDBC catalyst immersed in water for a long time. The long-term deactivations of the Pt/SDBC catalyst were evaluated quantitatively by mathematical models. The simple mathematical models were presented to evaluate the uniform poisoning and shell progressive poisoning to be occurred simultaneously during the hydrogen isotope exchange between hydrogen gas and liquid water in the Liquid Phase Catalytic Exchange(LPCE) column. The uniform poisoning was well characterized by a time on stream theory and then the deactivation parameters were determined from the experimental performance data. The impurity poisoning was derived by a shell progressive model with two-layer mass transfer. The water vapor condensation was a main cause of the reversible uniform poisoning for the Pt/SDBC catalyst. The values of the decay rate constant (K d ) and order of the decay reaction(m) were of 2 and 4, respectively, based on the experimental data. It indicated that the decay might be attributable to pore mouth poisoning. From the long-term stability of the catalyst immersed in water, there was no intrinsic decay of catalyst activity due to water logging to the catalyst. The activity decreased by only 7% over 18 months, which was equivalent to a catalyst half-life longer than 15 years. On the basis of the above deactivation parameters, the values for k c /k co with Thiele modulus=20 after 3 years and 10 years of operation were expected about 19% and 15% of the initial activity, respectively, while the values for k c /k co with Thiele modulus=100 were of about 22% and 18%, respectively

  7. Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry.

    Science.gov (United States)

    Tobler, S A; Sherman, N E; Fernandez, E J

    We utilized electrospray ionization mass spectrometry (ESI-MS) and hydrogen-deuterium exchange (HX) to detect unfolding of hen egg white lysozyme during salt-induced precipitation. Deuterated lysozyme was dissolved in protonated buffer at pH 2.16 and precipitated with ammonium sulfate, sodium chloride, and potassium thiocyanate. ESI-MS was used to detect mass differences in lysozyme due to the loss of deuterons for solvent protons, providing insight on the conformational history of the protein during the labeling experiment. Precipitation with ammonium sulfate and sodium chloride did not unfold lysozyme, consistent with the known stabilizing effects of kosmotropic salts. Potassium thiocyanate, an aggressive chaotrope, was an effective precipitant at 0.2 M, but also disrupted lysozyme structure and caused the formation of precipitate fractions that did not readily redissolve into aqueous solution without the use of a chemical denaturant. Precipitation with 1.0 M thiocyanate resulted in faster rates of unfolding and larger amounts of the insoluble precipitate. The unfolding kinetics were biphasic, exhibiting a slow phase after a few hours that presumably reflected a smaller propensity for lysozyme to unfold in the precipitated state. Bimodal mass distributions in the ESI-MS spectra for the thiocyanate precipitates indicate two states for lysozyme in this system, a native and a molten globule-like partially unfolded state. ESI-MS analysis of the insoluble precipitates indicated that they consisted primarily of protein molecules that had unfolded. Investigation of the HX behavior of lysozyme in a KSCN solution at low protein concentrations confirmed the destabilizing effect of the salt on the protein structure, even when there was almost no solid phase present. The HX/ESI-MS results provide insight into the mechanism combining precipitation and denaturation for such a system, both in terms of obtaining quantitative kinetic and stability information and the

  8. Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8.

    Science.gov (United States)

    Xu, Hua; Chen, Rongji; Ghishan, Fayez K

    2005-07-01

    Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na(+) absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.

  9. Nepenthesin from monkey cups for hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Rey, Martial; Yang, Menglin; Burns, Kyle M; Yu, Yaping; Lees-Miller, Susan P; Schriemer, David C

    2013-02-01

    Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners.

  10. Platform Dependencies in Bottom-up Hydrogen/Deuterium Exchange Mass Spectrometry*

    Science.gov (United States)

    Burns, Kyle M.; Rey, Martial; Baker, Charles A. H.; Schriemer, David C.

    2013-01-01

    Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. The bottom-up approach uses protein digestion to localize deuteration to higher resolution, and the essential measurement involves centroid mass determinations on a very large set of peptides. In the course of evaluating systems for various projects, we established two (HDX-MS) platforms that consisted of a FT-MS and a high-resolution QTOF mass spectrometer, each with matched front-end fluidic systems. Digests of proteins spanning a 20–110 kDa range were deuterated to equilibrium, and figures-of-merit for a typical bottom-up (HDX-MS) experiment were compared for each platform. The Orbitrap Velos identified 64% more peptides than the 5600 QTOF, with a 42% overlap between the two systems, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the QTOF, depending on the Orbitrap resolution setting. However, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate, because of destructive interference arising from mismatches in elemental mass defects. This is shown through the analysis of the peptides common to both platforms, where deuteration values can be as low as 35% of the expected values, depending on FT-MS resolution, peptide length and charge state. These findings are supported by simulations of Orbitrap transients, and highlight that caution should be exercised in deriving centroid mass values from FT transients that do not support baseline separation of the full isotopic composition. PMID:23197788

  11. Activation of ClpP protease by ADEP antibiotics: insights from hydrogen exchange mass spectrometry.

    Science.gov (United States)

    Sowole, Modupeola A; Alexopoulos, John A; Cheng, Yi-Qiang; Ortega, Joaquin; Konermann, Lars

    2013-11-15

    The bacterial protease ClpP consists of 14 subunits that assemble into two stacked heptameric rings. The central degradation chamber can be accessed via axial pores. In free ClpP, these pores are obstructed by the N-terminal regions of the seven subunits at either end of the barrel. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind in hydrophobic clefts surrounding the pore region, causing the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. Recently published X-ray structures yielded conflicting models regarding the conformation adopted by the N-terminal regions in the open state. Here, we use hydrogen/deuterium exchange (HDX) mass spectrometry to obtain complementary insights into the ClpP behavior with and without ADEP1. Ligand binding causes rigidification of the equatorial belt, accompanied by destabilization in the vicinity of the binding clefts. The N-terminal regions undergo rapid deuteration with only minor changes after ADEP1 binding, revealing a lack of stable H-bonding. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a "hydrophobic plug" causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way, ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore. This proposed mechanism helps reconcile the conflicting models that had been put forward earlier. © 2013.

  12. Platform dependencies in bottom-up hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Burns, Kyle M; Rey, Martial; Baker, Charles A H; Schriemer, David C

    2013-02-01

    Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. The bottom-up approach uses protein digestion to localize deuteration to higher resolution, and the essential measurement involves centroid mass determinations on a very large set of peptides. In the course of evaluating systems for various projects, we established two (HDX-MS) platforms that consisted of a FT-MS and a high-resolution QTOF mass spectrometer, each with matched front-end fluidic systems. Digests of proteins spanning a 20-110 kDa range were deuterated to equilibrium, and figures-of-merit for a typical bottom-up (HDX-MS) experiment were compared for each platform. The Orbitrap Velos identified 64% more peptides than the 5600 QTOF, with a 42% overlap between the two systems, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the QTOF, depending on the Orbitrap resolution setting. However, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate, because of destructive interference arising from mismatches in elemental mass defects. This is shown through the analysis of the peptides common to both platforms, where deuteration values can be as low as 35% of the expected values, depending on FT-MS resolution, peptide length and charge state. These findings are supported by simulations of Orbitrap transients, and highlight that caution should be exercised in deriving centroid mass values from FT transients that do not support baseline separation of the full isotopic composition.

  13. Kinetics of Exchange Between Zero-, One-, and Two-Hydrogen-Bonded States of Methyl and Ethyl Acetate in Methanol

    Science.gov (United States)

    Chuntonov, Lev; Pazos, Ileana M.; Ma, Jianqiang; Gai, Feng

    2015-01-01

    It has recently been shown that the ester carbonyl stretching vibration can be used as a sensitive probe of local electrostatic field in molecular systems. To further characterize this vibrational probe and extend its potential applications, we studied the kinetics of chemical exchange between differently hydrogen-bonded (H-bonded) ester carbonyl groups of methyl acetate (MA) and ethyl acetate (EA) in methanol. We found that while both MA and EA can form zero, one, or two H-bonds with the solvent, the population of the 2hb state in MA is significantly smaller than that in EA. Using a combination of linear and non-linear infrared measurements and numerical simulations, we further determined the rate constants for the exchange between these differently H-bonded states. We found that for MA the chemical exchange reaction between the two dominant states (i.e., 0hb and 1hb states) has a relaxation rate constant of 0.14 ps−1, whereas for EA the three-state chemical exchange reaction occurs in a predominantly sequential manner with the following relaxation rate constants: 0.11 ps−1 for exchange between 0hb and 1hb states, 0.12 ps−1 for exchange between 1hb and 2hb states. PMID:25738661

  14. Method for enriching and separating heavy hydrogen isotopes from substance streams containing such isotopes by means of isotope exchange

    International Nuclear Information System (INIS)

    Knochel, A.; Eggers, I.; Klatte, B.; Wilken, R. D.

    1985-01-01

    A process for enriching and separating heavy hydrogen isotopes having a heavy hydrogen cation (deuterium and/or tritium) from substance streams containing them, wherein the respectively present hydrogen isotopes are exchanged in chemical equilibria. A protic, acid solution containing deuterium and/or tritium is brought into contact with a value material from the group of open-chained polyethers or aminopolyethers, macro-monocyclic or macro-polycyclic polyethers, macro-monocyclic or macro-polycyclic amino polyethers, and mixtures of these values, in their free or proton salt form to form a reaction product of the heavy hydrogen cation with the value or value salt and bring about enrichment of deuterium and/or tritium in the reaction product. The reaction product containing the value or value salt is separated from the solution. The separated reaction product is treated to release the hydrogen isotope(s) to be enriched in the form of deuterium oxide (HDO) and/or tritium oxide (HTO) by regenerating the value or its salt, respectively. The regenerated value is returned for reuse

  15. Improvement of thermal exchange between feedstock and effluent in a hydrocarbon processing unit under hydrogen atmosphere by partial recycling of the product

    Energy Technology Data Exchange (ETDEWEB)

    Orieux, A.

    1990-01-19

    Heat exchange is improved in light naphta hydroisomerization and catalytic reforming by recirculation of a part of the product in the thermal exchange zone at a temperature higher than the dew point of the effluent under hydrogen atmosphere and preferentially as a temperature lower than the temperature of the recycled product.

  16. Imidazole C-2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with MALDI Mass Spectrometry

    Science.gov (United States)

    Hayashi, Naoka; Kuyama, Hiroki; Nakajima, Chihiro; Kawahara, Kazuki; Miyagi, Masaru; Nishimura, Osamu; Matsuo, Hisayuki; Nakazawa, Takashi

    2015-01-01

    We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine Cε1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second order with respect to the concentrations of the imidazolium cation and OD− in D2O. The second-order rate constant (k2) of this reaction was calculated from the pH-dependency of the pseudo-first-order rate constant (kφ) obtained from the change of average mass ΔMr (0 ≤ ΔMr exchange rate in terms of log(k2max/k2) representing the deviation of k2 from k2max. In the catalytic site of bovine ribonuclease A, His12 showed much larger change in log(k2max/k2) compared with His119 upon binding with cytidine 3′-monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, there is a need of considering the hydrogen bonds of the imidazole group with non-dissociable groups to interpret an extremely slow H/D exchange rate of His48 in partially solvent-exposed situation. PMID:24606199

  17. Surface modification of a proton exchange membrane and hydrogen storage in a metal hydride for fuel cells

    Science.gov (United States)

    Andrews, Lisa

    Interest in fuel cell technology is rising as a result of the need for more affordable and available fuel sources. Proton exchange membrane fuel cells involve the catalysis of a fuel to release protons and electrons. It requires the use of a polymer electrolyte membrane to transfer protons through the cell, while the electrons pass through an external circuit, producing electricity. The surface modification of the polymer, NafionRTM, commonly researched as a proton exchange membrane, may improve efficiency of a fuel cell. Surface modification can change the chemistry of the surface of a polymer while maintaining bulk properties. Plasma modification techniques such as microwave discharge of an argon and oxygen gas mixture as well as vacuum-ultraviolet (VUV) photolysis may cause favorable chemical and physical changes on the surface of Nafion for improved fuel cell function. A possible increase in hydrophilicity as a result of microwave discharge experiments may increase proton conductivity. Grafting of acrylic acid from the surface of modified Nafion may decrease the permeation of methanol in a direct methanol fuel cell, a process which can decrease efficiency. Modification of the surface of Nafion samples were carried out using: 1) An indirect Ar/O2 gas mixture plasma investigating the reaction of oxygen radicals with the surface, 2) A direct Ar/O2 gas mixture plasma investigating the reaction of oxygen radicals and VUV radiation with the surface and, 3) VUV photolysis investigating exclusively the interaction of VUV radiation with the surface and any possible oxidation upon exposure to air. Acrylic acid was grafted from the VUV photolysed Nafion samples. All treated surfaces were analyzed using X-ray photoelectron spectroscopy (XPS). Fourier transform infrared spectroscopy (FTIR) was used to analyze the grafted Nafion samples. Scanning electron microscopy (SEM) and contact angle measurements were used to analyze experiments 2 and 3. Using hydrogen as fuel is a

  18. Deuterium-hydrogen exchange of oxygen-containing heterocyclic compounds with D2O

    International Nuclear Information System (INIS)

    Dedov, A.G.; Brezhnev, L.YU.; Karakhanov, Eh.A.

    1983-01-01

    H-D-exchange of furan series compounds with D 2 O in the presence of activated carbon and platinum has been studied. In the case of Pt the compounds studied are arranged according to the degree of deuteration in the following series: benzofuran > sylvan > furan. In the case of carbon the picture is as follows: 2, 3-dihydrofuran > sylvan >> benzofuran. It is shown that, in contrast with Pt, the H-D-exchange on carbon follows the ion exchange mechanism

  19. Maximum Potential Hydrogen Gas Retention in the sRF Resin Ion Exchange Column for the LAWPS Process

    Energy Technology Data Exchange (ETDEWEB)

    Gauglitz, Phillip A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Wells, Beric E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Bottenus, Courtney LH [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Schonewill, Philip P. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2018-01-22

    The Low-Activity Waste Pretreatment System (LAWPS) is being developed to provide treated supernatant liquid from the Hanford tank farms directly to the Low-Activity Waste (LAW) Vitrification Facility at the Hanford Tank Waste Treatment and Immobilization Plant. The design and development of the LAWPS is being conducted by Washington River Protection Solutions, LLC. A key process in LAWPS is the removal of radioactive Cs in ion exchange (IX) columns filled with spherical resorcinol-formaldehyde (sRF) resin. One accident scenario being evaluated is the loss of liquid flow through the sRF resin bed after it has been loaded with radioactive Cs and hydrogen gas is being generated by radiolysis. In normal operations, the generated hydrogen is expected to remain dissolved in the liquid and be continuously removed by liquid flow. For an accident scenario with a loss of flow, hydrogen gas can be retained within the IX column both in the sRF resin and below the bottom screen that supports the resin within the column. The purpose of this report is to summarize calculations that estimate the upper-bound volume of hydrogen gas that can be retained in the column and potentially be released to the headspace of the IX column or to process equipment connected to the IX column and, thus, pose a flammability hazard.

  20. Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative tris(2,4,6-trimethoxyphenyl) phosphonium: implications for the aspartic acid cleavage mechanism.

    Science.gov (United States)

    Herrmann, Kristin A; Wysocki, Vicki H; Vorpagel, Erich R

    2005-07-01

    Aspartic acid (Asp)-containing peptides with the fixed charge derivative tris(2,4,6trimethoxyphenyl) phosphonium (tTMP-P+) were explored computationally and experimentally by hydrogen/deuterium (H/D) exchange and by fragmentation studies to probe the phenomenon of selective cleavage C-terminal to Asp in the absence of a "mobile" proton. Ab initio modeling of the tTMP-P+ electrostatic potential shows that the positive charge is distributed on the phosphonium group and therefore is not initiating or directing fragmentation as would a "mobile" proton. Geometry optimizations and vibrational analyses of different Asp conformations show that the Asp structure with a hydrogen bond between the side-chain hydroxy and backbone carbonyl lies 2.8 kcal/mol above the lowest energy conformer. In reactions with D2O, the phosphonium-derived doubly charged peptide (H+)P+LDIFSDF rapidly exchanges all 12 of its exchangeable hydrogens for deuterium and also displays a nonexchanging population. With no added proton, P+LDIFSDF exchanges a maximum of 4 of 11 exchangeable hydrogens for deuterium. No exchange is observed when all acidic groups are converted to the corresponding methyl esters. Together, these H/D exchange results indicate that the acidic hydrogens are "mobile locally" because they are able to participate in exchange even in the absence of an added proton. Fragmentation of two distinct (H+)P+LDIFSDF ion populations shows that the nonexchanging population displays selective cleavage, whereas the exchanging population fragments more evenly across the peptide backbone. This result indicates that H/D exchange can sometimes distinguish between and provide a means of separation of different protonation motifs and that these protonation motifs can have an effect on the fragmentation.

  1. Catalytic hydrogen/oxygen reaction assisted the proton exchange membrane fuel cell (PEMFC) startup at subzero temperature

    Science.gov (United States)

    Sun, Shucheng; Yu, Hongmei; Hou, Junbo; Shao, Zhigang; Yi, Baolian; Ming, Pingwen; Hou, Zhongjun

    Fuel cells for automobile application need to operate in a wide temperature range including freezing temperature. However, the rapid startup of a proton exchange membrane fuel cell (PEMFC) at subfreezing temperature, e.g., -20 °C, is very difficult. A cold-start procedure was developed, which made hydrogen and oxygen react to heat the fuel cell considering that the FC flow channel was the characteristic of microchannel reactor. The effect of hydrogen and oxygen reaction on fuel cell performance at ambient temperature was also investigated. The electrochemical characterizations such as I- V plot and cyclic voltammetry (CV) were performed. The heat generated rate for either the single cell or the stack was calculated. The results showed that the heat generated rate was proportional to the gas flow rate when H 2 concentration and the active area were constant. The fuel cell temperature rose rapidly and steadily by controlling gas flow rate.

  2. Gas-phase fragmentation of peptides to increase the spatial resolution of the Hydrogen Exchange Mass Spectrometry experiment

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Rand, Kasper Dyrberg

    2016-01-01

    are produced after precursor ion selection and thus do not add complexity to the LC-MS analysis. The key to obtaining optimal spatial resolution in a hydrogen exchange mass spectrometry (HX-MS) experiment is the fragmentation efficiency. This chapter discusses common fragmentation techniques like collision......-induced dissociation (CID) occur with complete Hydrogen-deuterium (H/D) scrambling, while other techniques that induce dissociation on a faster timescale through radical-based fragmentation channels, like electron-capture dissociation (ECD) and electron-transfer dissociation (ETD), occur inherently without H......Generation of overlapping peptides in solution via multiple proteases requires a very high peak capacity for the LC-MS analysis to minimize signal overlap. An inherent advantage of a gas-phase fragmentation step is that the additional gas-phase fragment ions used to sublocalize deuterium...

  3. Preparation and hydrogen-deuterium exchange of alkyl and hydride bis(trimethylsilyl)amido derivatives of the actinide elements

    International Nuclear Information System (INIS)

    Simpson, S.J.; Turner, H.W.; Andersen, R.A.

    1981-01-01

    The monomeric, hydrocarbon-soluble monohydrides and monodeuterides of the actinide metals (thorium or uranium) of the type HM[N(SiMe 3 ) 2 ] 3 have been prepared. Their reaction chemistry - n-BuLi followed by MeBr yields MeM[N(SiMe 3 ) 2 ] 3 and borane in tetrahydrofuran yields BH 4 M[N(SiMe 3 ) 2 ] 3 - suggests that the hydrogen atom is hydridic. Pyrolysis of the hydrides yields the novel, four-membered ring metallacycle [(Me 3 Si) 2 N] 2 MCH 2 Si(Me) 2 NSiMe 3 where M is Th or U. These metallacycles are the key intermediates in the hydrogen-deuterium exchange reaction that yields ([CD 3 ) 3 Si] 2 N) 3 MD

  4. Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Houde, Damian; Esmail Nazari, Zeinab; Bou-Assaf, George M

    2016-01-01

    for these phenomena can be due to short range electrostatic and/or hydrophobic protein-protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, "traditional" continuous dilution labeling HDX......-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied...

  5. Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Huang, Richard Y.-C.; Iacob, Roxana E.; Krystek, Stanley R.; Jin, Mi; Wei, Hui; Tao, Li; Das, Tapan K.; Tymiak, Adrienne A.; Engen, John R.; Chen, Guodong

    2017-05-01

    Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.

  6. Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution

    Science.gov (United States)

    Zhu, Shaolong; Campbell, J. Larry; Chernushevich, Igor; Le Blanc, J. C. Yves; Wilson, Derek J.

    2016-06-01

    Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development.

  7. Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications.

    Science.gov (United States)

    Wei, Hui; Mo, Jingjie; Tao, Li; Russell, Reb J; Tymiak, Adrienne A; Chen, Guodong; Iacob, Roxana E; Engen, John R

    2014-01-01

    The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this review, HDX-MS based workflows designed for protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS in protein therapeutics characterization are also described. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

    DEFF Research Database (Denmark)

    Esmail Nazari, Zeinab; van de Weert, Marco; Bou-Assaf, George

    2016-01-01

    Hydrogen Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) has become an established method for analysis of protein higher-order structure. Here, we use HDX-MS methodology based on manual Solid-Phase Extraction (SPE) to allow fast and simplified conformational analysis of proteins under...... pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In Mode 1, we used DMSO-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable...... in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that the SPE-HDX-MS system can...

  9. Using a Potassium Acetate Solution for Cooling High Pressure Hydrogen in a Prototype Heat Exchanger

    DEFF Research Database (Denmark)

    Rothuizen, Erasmus Damgaard; Abel, M.; Rokni, Masoud

    2011-01-01

    is to be delivered at high pressure a heat exchanger was designed and constructed. The paper presents a detailed study of construction of the heat exchanger which has been tested and compared to theory to predict and verify its performance. The method presented by Nellis and Klein for laminar flow in annulus tubes...

  10. Hydrogen-deuterium exchange reactions of carbanions with D2O in the gas phase

    International Nuclear Information System (INIS)

    Stewart, J.H.; Shapiro, R.H.; DePuy, C.H.; Bierbaum, V.M.

    1977-01-01

    Using the flowing afterglow technique, we have observed that carbanions participate in sequential deuterium exchange reactions with D 2 O in the gas phase. The extent of exchange is reported for 32 carbanions and the mechanism of the reaction is discussed. The usefulness of this phenomenon as a probe of the acidity and structure of anions is described

  11. Characterizing the dynamics of α-synuclein oligomers using hydrogen/deuterium exchange monitored by mass spectrometry.

    Science.gov (United States)

    Mysling, Simon; Betzer, Cristine; Jensen, Poul H; Jorgensen, Thomas J D

    2013-12-23

    Soluble oligomers formed by α-synuclein (αSN) are suspected to play a central role in neuronal cell death during Parkinson's disease. While studies have probed the surface structure of these oligomers, little is known about the backbone dynamics of αSN when they form soluble oligomers. Using hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS), we have analyzed the structural dynamics of soluble αSN oligomers. The analyzed oligomers were metastable, slowly dissociating to monomers over a period of 21 days, after excess monomer had been removed. The C-terminal region of αSN (residues 94-140) underwent isotopic exchange very rapidly, demonstrating a highly dynamic region in the oligomeric state. Three regions (residues 4-17, 39-54, and 70-89) were strongly protected against isotopic exchange in the oligomers, indicating the presence of a stable hydrogen-bonded or solvent-shielded structure. The protected regions were interspersed by two somewhat more dynamic regions (residues 18-38 and 55-70). In the oligomeric state, the isotopic exchange pattern of the region of residues 35-95 of αSN corresponded well with previous nuclear magnetic resonance and electron paramagnetic resonance analyses performed on αSN fibrils and indicated a possible zipperlike maturation mechanism for αSN aggregates. We find the protected N-terminus (residues 4-17) to be of particular interest, as this region has previously been observed to be highly dynamic for both monomeric and fibrillar αSN. This region has mainly been described in relation to membrane binding of αSN, and structuring may be important in relation to disease.

  12. Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

    Science.gov (United States)

    Keppel, Theodore R.; Weis, David D.

    2015-04-01

    Measurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such proteins undergo coupled binding and folding. The present work describes an approach to measure residual structure in disordered proteins using millisecond hydrogen/deuterium (H/D) exchange in a conventional bottom-up peptide-based workflow. We used the exchange mid-point, relative to a totally deuterated control, to quantify the rate of H/D exchange in each peptide. A weighted residue-by-residue average of these midpoints was used to map the extent of residual structure at near single-residue resolution. We validated this approach both by simulating a disordered protein and experimentally using the p300 binding domain of ACTR, a model disordered protein already well-characterized by other approaches. Secondary structure elements mapped in the present work are in good agreement with prior nuclear magnetic resonance measurements. The new approach was somewhat limited by a loss of spatial resolution and subject to artifacts because of heterogeneities in intrinsic exchange. Approaches to correct these limitations are discussed.

  13. Development of polarized negative hydrogen ion source with resonant charge-exchange plasma ionizer

    Science.gov (United States)

    Belov, A. S.; Esin, S. K.; Netchaeva, L. P.; Turbabin, A. V.; Vasil'Ev, G. A.

    2001-06-01

    Polarized negative hydrogen ion beam with peak current of 2.5 mA has been obtained from an atomic beam-type polarized ion source of Institute for Nuclear Research, Moscow. The intensity improvement has been achieved due to increase of efficiency of conversion of polarized hydrogen atoms into polarized negative ions. New converter for production of deuterium plasma with high density of unpolarized negative ions is described. Limitations of the method and possible improvements are discussed. .

  14. Polymer amide as an early topology.

    Science.gov (United States)

    McGeoch, Julie E M; McGeoch, Malcolm W

    2014-01-01

    Hydrophobic polymer amide (HPA) could have been one of the first normal density materials to accrete in space. We present ab initio calculations of the energetics of amino acid polymerization via gas phase collisions. The initial hydrogen-bonded di-peptide is sufficiently stable to proceed in many cases via a transition state into a di-peptide with an associated bound water molecule of condensation. The energetics of polymerization are only favorable when the water remains bound. Further polymerization leads to a hydrophobic surface that is phase-separated from, but hydrogen bonded to, a small bulk water complex. The kinetics of the collision and subsequent polymerization are discussed for the low-density conditions of a molecular cloud. This polymer in the gas phase has the properties to make a topology, viz. hydrophobicity allowing phase separation from bulk water, capability to withstand large temperature ranges, versatility of form and charge separation. Its flexible tetrahedral carbon atoms that alternate with more rigid amide groups allow it to deform and reform in hazardous conditions and its density of hydrogen bonds provides adhesion that would support accretion to it of silicon and metal elements to form a stellar dust material.

  15. Polymer amide as an early topology.

    Directory of Open Access Journals (Sweden)

    Julie E M McGeoch

    Full Text Available Hydrophobic polymer amide (HPA could have been one of the first normal density materials to accrete in space. We present ab initio calculations of the energetics of amino acid polymerization via gas phase collisions. The initial hydrogen-bonded di-peptide is sufficiently stable to proceed in many cases via a transition state into a di-peptide with an associated bound water molecule of condensation. The energetics of polymerization are only favorable when the water remains bound. Further polymerization leads to a hydrophobic surface that is phase-separated from, but hydrogen bonded to, a small bulk water complex. The kinetics of the collision and subsequent polymerization are discussed for the low-density conditions of a molecular cloud. This polymer in the gas phase has the properties to make a topology, viz. hydrophobicity allowing phase separation from bulk water, capability to withstand large temperature ranges, versatility of form and charge separation. Its flexible tetrahedral carbon atoms that alternate with more rigid amide groups allow it to deform and reform in hazardous conditions and its density of hydrogen bonds provides adhesion that would support accretion to it of silicon and metal elements to form a stellar dust material.

  16. Amides in Nature and Biocatalysis.

    Science.gov (United States)

    Pitzer, Julia; Steiner, Kerstin

    2016-10-10

    Amides are widespread in biologically active compounds with a broad range of applications in biotechnology, agriculture and medicine. Therefore, as alternative to chemical synthesis the biocatalytic amide synthesis is a very interesting field of research. As usual, Nature can serve as guide in the quest for novel biocatalysts. Several mechanisms for carboxylate activation involving mainly acyl-adenylate, acyl-phosphate or acyl-enzyme intermediates have been discovered, but also completely different pathways to amides are found. In addition to ribosomes, selected enzymes of almost all main enzyme classes are able to synthesize amides. In this review we give an overview about amide synthesis in Nature, as well as biotechnological applications of these enzymes. Moreover, several examples of biocatalytic amide synthesis are given. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. New developments in protein structure-function analysis by MS and use of hydrogen-deuterium exchange microfluidics.

    Science.gov (United States)

    Landreh, Michael; Astorga-Wells, Juan; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-10-01

    The study of protein structure and function has evolved to become a leading discipline in the biophysical sciences. Although it is not yet possible to determine 3D protein structures from MS data alone, multiple MS-based techniques can be combined to obtain structural and functional data that are complementary to classical protein structure information obtained from NMR or X-ray crystallography. Monitoring gas-phase interactions of noncovalent complexes yields information on binding constants, complex stability, and the nature of interactions. Ion mobility MS and chemical crosslinking strategies can be applied to probe the architecture of macromolecular assemblies and protein-ligand complexes. MS analysis of hydrogen-deuterium exchange can be used to determine the localization of secondary structure elements, binding sites and conformational dynamics of proteins in solution. This minireview focuses first on new strategies that combine these techniques to gain insights into protein structure and function. Using one such strategy, we then demonstrate how a novel hydrogen-deuterium exchange microfluidics tool can be used online with an ESI mass spectrometer to monitor regional accessibility in a peptide, as exemplified with amyloid-β peptide 1-40. © 2011 The Authors Journal compilation © 2011 FEBS.

  18. Polymerization dependence of the reactivity of polyacrylamide observed with hydrogen-isotope exchange reaction in a liquid-solid system

    International Nuclear Information System (INIS)

    Imaizumi, Hiroshi; Ishii, Tatsuya

    1996-01-01

    The tritium (T) labeled polyacrylamide (abbreviated PAAm(T) below) was synthesized using the hydrogen-isotope exchange reaction (gas-solid reaction) between HTO vapor and PAAm. The degree of the polymerization of PAAm used was 2800 (and 80000) (abbreviated PAAm 2800 (and PAAm 80000 ) below). Using the PAAm(T) thus obtained, the hydrogen-isotope exchange reaction (liquid-solid reaction) between PAAm(T) and each liquid organic material has been observed at the temperature range of 50 to 90degC. Applying the A''-McKay plot method to the data thus obtained, the rate constant (k) for the reaction was obtained. Including k obtained previously, the value of k thus obtained were compared with each other. The following six items have consequently been confirmed. The reactivity of PAAm 80000 is larger than that of PAAm 2800 . PAAm 2800 is about 0.4 times PVA 2900 in reactivity. The temperature dependence of the reactivity of PAAm 2800 is about 6 times that of PVA-2 900 . The reactivity of these three compounds for several liquid organic materials can roughly be expressed as follows: (PVA 2900 ):(PAAm 80000 ):(PAAm 2800 )=1:1:0.3. It is possible to use PAAm as a solid material in the liquid-solid reaction (instead of PVA). The method used in this work can be useful to clarify the reactivity of a certain material, and to obtain the data for the prevention of tritium-contamination. (author)

  19. Gastrointestinal Inhibition of Sodium-Hydrogen Exchanger 3 Reduces Phosphorus Absorption and Protects against Vascular Calcification in CKD.

    Science.gov (United States)

    Labonté, Eric D; Carreras, Christopher W; Leadbetter, Michael R; Kozuka, Kenji; Kohler, Jill; Koo-McCoy, Samantha; He, Limin; Dy, Edward; Black, Deborah; Zhong, Ziyang; Langsetmo, Ingrid; Spencer, Andrew G; Bell, Noah; Deshpande, Desiree; Navre, Marc; Lewis, Jason G; Jacobs, Jeffrey W; Charmot, Dominique

    2015-05-01

    In CKD, phosphate retention arising from diminished GFR is a key early step in a pathologic cascade leading to hyperthyroidism, metabolic bone disease, vascular calcification, and cardiovascular mortality. Tenapanor, a minimally systemically available inhibitor of the intestinal sodium-hydrogen exchanger 3, is being evaluated in clinical trials for its potential to (1) lower gastrointestinal sodium absorption, (2) improve fluid overload-related symptoms, such as hypertension and proteinuria, in patients with CKD, and (3) reduce interdialytic weight gain and intradialytic hypotension in ESRD. Here, we report the effects of tenapanor on dietary phosphorous absorption. Oral administration of tenapanor or other intestinal sodium-hydrogen exchanger 3 inhibitors increased fecal phosphorus, decreased urine phosphorus excretion, and reduced [(33)P]orthophosphate uptake in rats. In a rat model of CKD and vascular calcification, tenapanor reduced sodium and phosphorus absorption and significantly decreased ectopic calcification, serum creatinine and serum phosphorus levels, circulating phosphaturic hormone fibroblast growth factor-23 levels, and heart mass. These results indicate that tenapanor is an effective inhibitor of dietary phosphorus absorption and suggest a new approach to phosphate management in renal disease and associated mineral disorders. Copyright © 2015 by the American Society of Nephrology.

  20. Abstraction and exchange contributions to the rate constant of muonium + hydrogen chloride reaction

    International Nuclear Information System (INIS)

    Lagana, A.; Ciccarelli, L.

    1987-01-01

    Quantum collinear rate constants for the abstraction and the exchange channels of the Mu + HCl reaction have been calculated in order to have an estimate of the relative efficiency of the two processes in promoting reactivity for this system. (orig.)

  1. Abstraction and exchange contributions to the rate constant of muonium+hydrogen chloride reaction

    Science.gov (United States)

    Laganà, A.; Ciccarelli, L.

    1987-02-01

    Quantum collinear rate constants for the abstraction and the exchange channels of the Mu+HCl reaction have been calculated in order to have an estimate of the relative efficiency of the two processes in promoting reactivity for this system.

  2. Development of an analytical diffusion model for modeling hydrogen isotope exchange

    Science.gov (United States)

    Barton, J. L.; Wang, Y. Q.; Doerner, R. P.; Tynan, G. R.

    2015-08-01

    We create a model for H retention depth profiles in W and subsequently model how this profile changes after isotope exchange. This is accomplished by calculating how trapping defects in W accumulate D (or H) inventory as W is being exposed to plasma. Each trapping site is characterized by a trapping rate and a release rate, where the only free parameters are the distribution of these trapping sites in the material. The filled trap concentrations for each trap type are modeled as a diffusion process because post-mortem deuterium depth profiles indicate that traps are filled well beyond the ion implantation zone (2-5 nm). Using this retention model, an isotope exchange rate is formulated. The retention model and isotope exchange rate are compared to low temperature isotope exchange experiments in tungsten with good agreement. The limitations of the current model highlight important physics and motivate future work.

  3. Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Tonelli, Marco; Singarapu, Kiran K. [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States); Makino, Shin-ichi; Sahu, Sarata C.; Matsubara, Yuko [University of Wisconsin-Madison, Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry (United States); Endo, Yaeta [Ehime University, Cell-Free Science and Technology Research Center (Japan); Kainosho, Masatsune [Tokyo Metropolitan University, Center for Priority Areas (Japan); Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States)

    2011-12-15

    Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H{sub 2}O, exchange reactions can lead to contamination of {sup 2}H sites by {sup 1}H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing {sup 1}H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM l-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-{sup 2}H, {sup 15}N]-chlorella ubiquitin without and with added inhibitors, and [U-{sup 15}N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-{sup 13}C, {sup 15}N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at C{sup {alpha}} sites, with the exception of Gly, and at C{sup {beta}} sites of Ala. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Asn-H{sup {beta}}, Asp-H{sup {beta}}, Gln-H{sup {gamma}}, Glu-H{sup {gamma}}, and Lys-H{sup {epsilon}}. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of

  4. Hydrogenation

    Energy Technology Data Exchange (ETDEWEB)

    Pier, M.

    1943-02-19

    A transcript is presented of a speech on the history of the development of hydrogenation of coal and tar. Apparently the talk had been accompanied by the showing of photographic slides, but none of the pictures were included with the report. In giving the history, Dr. Pier mentioned the dependence of much of the development of hydrogenation upon previous development in the related areas of ammonia and methanol syntheses, but he also pointed out several ways in which equipment appropriate for hydrogenation differed considerably from that used for ammonia and methanol. Dr. Pier discussed the difficulties encountered with residue processing, design of the reaction ovens, manufacture of ovens and preheaters, heating of reaction mixtures, development of steels, and development of compressor pumps. He described in some detail his own involvement in the development of the process. In addition, he discussed the development of methods of testing gasolines and other fuels. Also he listed some important byproducts of hydrogenation, such as phenols and polycyclic aromatics, and he discussed the formation of iso-octane fuel from the butanes arising from hydrogenation. In connection with several kinds of equipment used in hydrogenation (whose pictures were being shown), Dr. Pier gave some of the design and operating data.

  5. Online Simultaneous Hydrogen/Deuterium Exchange of Multitarget Gas-Phase Molecules by Electrospray Ionization Mass Spectrometry Coupled with Gas Chromatography.

    Science.gov (United States)

    Jeong, Eun Sook; Cha, Eunju; Cha, Sangwon; Kim, Sunghwan; Oh, Han Bin; Kwon, Oh-Seung; Lee, Jaeick

    2017-11-21

    In this study, a hydrogen/deuterium (H/D) exchange method using gas chromatography-electrospray ionization/mass spectrometry (GC-ESI/MS) was first investigated as a novel tool for online H/D exchange of multitarget analytes. The GC and ESI source were combined with a homemade heated column transfer line. GC-ESI/MS-based H/D exchange occurs in an atmospheric pressure ion source as a result of reacting the gas-phase analyte eluted from GC with charged droplets of deuterium oxide infused as the ESI spray solvent. The consumption of the deuterated solvent at a flow rate of 2 μL min -1 was more economical than that in online H/D exchange methods reported to date. In-ESI-source H/D exchange by GC-ESI/MS was applied to 11 stimulants with secondary amino or hydroxyl groups. After H/D exchange, the spectra of the stimulants showed unexchanged, partially exchanged, and fully exchanged ions showing various degrees of exchange. The relative abundances corrected for naturally occurring isotopes of the fully exchanged ions of stimulants, except for etamivan, were in the range 24.3-85.5%. Methylephedrine and cyclazodone showed low H/D exchange efficiency under acidic, neutral, and basic spray solvent conditions and nonexchange for etamivan with an acidic phenolic OH group. The in-ESI-source H/D exchange efficiency by GC-ESI/MS was sufficient to determine the number of hydrogen by elucidation of fragmentation from the spectrum. Therefore, this online H/D exchange technique using GC-ESI/MS has potential as an alternative method for simultaneous H/D exchange of multitarget analytes.

  6. Information exchange on HTGR and nuclear hydrogen technology between JAEA and INET in 2009

    International Nuclear Information System (INIS)

    Fujimoto, Nozomu; Wang Hong

    2010-07-01

    The worldwide interests in the HTGR (High Temperature Gas-cooled Reactor) have been growing because the high temperature heat produced by the reactor can be utilized not only for efficient power generation but also for broad process heat applications, especially for thermo-chemical hydrogen production to fuel a prospective hydrogen economy in future. Presently only two HTGR reactors are operational in the world, including the HTTR (High Temperature Engineering Test Reactor) in Japan Atomic Energy Agency (JAEA) and the HTR-10 in the Institute of Nuclear and New Energy Technology (INET) of Tsinghua University in China. JAEA and INET have cooperated since 1986 in the field of HTGR development, particularly on the HTTR and HTR-10 projects. This report describes the cooperation activities on HTGR and nuclear hydrogen technology between JAEA and INET in 2009. (author)

  7. Information exchange on HTGR and nuclear hydrogen technology between JAEA and INET in 2008

    International Nuclear Information System (INIS)

    Fujimoto, Nozomu; Tachibana, Yukio; Sun Yuliang

    2009-07-01

    The worldwide interests in the HTGR (High Temperature Gas-cooled Reactor) have been growing because the high temperature heat produced by the reactor can be utilized not only for efficient power generation but also for broad process heat applications, especially for thermo-chemical hydrogen production to fuel a prospective hydrogen economy in future. Presently only two HTGR reactors are operational in the world, including the HTTR (High Temperature Engineering Test Reactor) in Japan Atomic Energy Agency (JAEA) and the HTR-10 in the Institute of Nuclear and New Energy Technology (INET) of Tsinghua University in China. JAEA and INET have cooperated since 1986 in the field of HTGR development, particularly on the HTTR and HTR-10 projects. This report describes the cooperation activities on HTGR and nuclear hydrogen technology between JAEA and INET in 2008. (author)

  8. Benchmarking exchange-correlation functionals for hydrogen at high pressures using quantum Monte Carlo

    Energy Technology Data Exchange (ETDEWEB)

    Clay, Raymond C. [Univ. of Illinois, Urbana, IL (United States); Mcminis, Jeremy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McMahon, Jeffrey M. [Univ. of Illinois, Urbana, IL (United States); Pierleoni, Carlo [Istituto Nazionale di Fisica Nucleare (INFN), L' aquila (Italy). Lab. Nazionali del Gran Sasso (INFN-LNGS); Ceperley, David M. [Univ. of Illinois, Urbana, IL (United States); Morales, Miguel A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2014-05-01

    The ab initio phase diagram of dense hydrogen is very sensitive to errors in the treatment of electronic correlation. Recently, it has been shown that the choice of the density functional has a large effect on the predicted location of both the liquid-liquid phase transition and the solid insulator-to-metal transition in dense hydrogen. To identify the most accurate functional for dense hydrogen applications, we systematically benchmark some of the most commonly used functionals using quantum Monte Carlo. By considering several measures of functional accuracy, we conclude that the van der Waals and hybrid functionals significantly outperform local density approximation and Perdew-Burke-Ernzerhof. We support these conclusions by analyzing the impact of functional choice on structural optimization in the molecular solid, and on the location of the liquid-liquid phase transition.

  9. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry*

    Science.gov (United States)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D.

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. PMID:25378534

  10. Hydrogen/deuterium exchange-protected oligomers populated during Aβ fibril formation correlate with neuronal cell death.

    Science.gov (United States)

    Serra-Vidal, Bernat; Pujadas, Lluís; Rossi, Daniela; Soriano, Eduardo; Madurga, Sergio; Carulla, Natàlia

    2014-11-21

    The aggregation of the amyloid-β peptide (Aβ) to form fibrils and plaques is strongly associated with Alzheimer's disease (AD). Although it is well established that this process generates neurotoxicity, it is also heterogeneous with a variety of species being formed during the conversion process. This heterogeneity makes it difficult to detect and characterize each of the aggregates formed, which precludes establishing the specific features responsible for the neurotoxicity observed. Here we use pulse-labeling hydrogen-deuterium exchange experiments analyzed by electrospray ionization mass spectrometry (PL-HDX-ESI-MS) to distinguish three ensembles populated during the aggregation of the 40 and 42 residue forms of the Aβ peptide, Aβ40 and Aβ42, on the basis of differences in their persistent structure. Noticeably, two of them are more abundant at the beginning and at the end of the lag phase and are therefore not detectable by conventional assays such as Thioflavin T (ThT). The ensembles populated at different stages of the aggregation process have a surprisingly consistent average degree of exchange, indicating that there are definite structural transitions between the different stages of aggregation. To determine whether an ensemble of species with a given hydrogen exchange pattern correlates with neurotoxicity, we combined PL-HDX-ESI-MS experiments with parallel measurements of the neurotoxicity of the samples under study. The results of this dual approach show that the maximum toxicity correlates with the ensemble comprising HDX protected oligomers, indicating that development of persistent structure within Aβ oligomers is a determinant of neurotoxicity.

  11. Investigating the interaction between the neonatal Fc receptor and monoclonal antibody variants by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG(1) and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Hydrogen/deuterium exchange mass spectrometry identifies two highly protected regions in recombinant full-length prion protein amyloid fibrils.

    Science.gov (United States)

    Nazabal, Alexis; Hornemann, Simone; Aguzzi, Adriano; Zenobi, Renato

    2009-06-01

    Understanding the structural basis that distinguishes the amyloid form of the prion protein from its monomeric homologue is of crucial importance to elucidate the mechanism of the lethal diseases related to this protein. Recently, an in vitro conversion system was established which reproduces the transition of recombinant prion protein PrP(23-230) from its native alpha-helical rich form into an aggregated amyloid beta-sheet rich form with physicochemical properties reminiscent to those of the disease-related isoform of the prion protein, PrPSc. To study the tertiary and quaternary structural organization within recombinant amyloid fibrils from mouse, mPrP(23-231)betaf; bovine, bPrP(23-230)betaf; and elk, ePrP(23-230)betaf; we utilized hydrogen/deuterium (H/D) exchange analyzed by matrix-assisted laser desorption/ionization (MALDI) and nano-electrospray (nano-ESI) mass spectrometry. No significant differences were found by measuring the deuterium exchange kinetics of the aggregated fibrillar forms for mPrP(23-231)betaf, bPrP(23-230)betaf and ePrP(23-230)betaf, indicating a similar overall structural organization of the fibrils from all three species. Next, we characterized the solvent accessibility for the soluble and fibrillar forms of the mouse prion protein by hydrogen exchange, pepsin proteolysis and nano-ESI ion trap mass spectrometry analysis. In its amyloid form, two highly protected regions of mPrP(23-231) comprising residues [24-98] and [182-212] were identified. The residues between the two highly protected stretches were found to be more solvent exposed, but less than in the soluble protein, and might therefore rather form part of a fibrillar interface. Copyright 2009 John Wiley & Sons, Ltd.

  13. Exchangeable hydrogen explains the pH of spodosol Oa horizons

    Science.gov (United States)

    Ross, D.S.; David, M.B.; Lawrence, G.B.; Bartlett, R.J.

    1996-01-01

    The chemistry of extremely acid Oa horizons does not conform to traditional pH, Al, and base saturation relationships. Results from two separate studies of northeastern U.S. forested soils were used to investigate relationships between pH in water or dilute salt solutions and other soil characteristics. In Oa horizons with pH below 4, soil pH in dilute CaCl2 solution was correlated with exchangeable H+ measured either by titration (r = -0.88, P = 0.0001, n = 142) or by electrode (r = -0.89, P = 0.0001, n = 45). Exchangeable H+ expressed as a percentage of the cation-exchange capacity (CEC) was linear with pH and showed similar slopes for data from both studies. For all samples, pHw = 4.21 - 1.80 x H+/CEC (R2 = 0.69, n = 194). The reciprocal of the H+/CEC ratio is base saturation with Al added to the bases. Because of the low pH, exchangeable Al does not appear to behave as an acid. Exchangeable H+ remains an operationally defined quantity because of the difficulty in separating exchange and hydrolysis reactions. In a variety of neutral-salt extractants, concentration of H+ were correlated with 0.1 M BaCl2-exchangeable H+ (r > 0.91, P = 0.0001, n = 26) regardless of the strength of the extract. Nine successive extractions with 0.33 mM CaCl2 removed more H+ than was removed by single batch extractions with either 1 M KCl or 0.1 M BaCl2 (average H+ of 70, 43, and 49 mmol kg-1, respectively for 26 samples). The data showed little difference in the chemical behavior of Oa horizons from a variety of geographical sites and vegetation types.

  14. Analysis of surface structure and hydrogen/deuterium exchange of colloidal silica suspension by contrast-variation small-angle neutron scattering.

    Science.gov (United States)

    Suzuki, Takuya; Endo, Hitoshi; Shibayama, Mitsuhiro

    2008-05-06

    The microscopic surface structure and hydrogen/deuterium exchange effect were investigated by contrast-variation small-angle neutron scattering (CV-SANS) for three different-sized amorphous colloidal silica aqueous suspensions. The results show that the fraction of hydrogen/deuterium exchange per nanoparticle, phiH/D, strongly depends on the size of silica nanoparticles. This finding supports that the hydrogen/deuterium exchange occurs exclusively within a finite surface layer of silica nanoparticles, while the inner component remained unchanged. Detailed analyses of the scattering intensity functions led to the estimation of (1) phiH/D and (2) the thickness of the surface layer as functions of the particle radius. The surface layer thickness was found to increase from 18 to 35 A with decreasing the particle radius from 165 to 71.2 A. The surface area per unit weight of silica estimated with the CV-SANS results are comparable to those reported in the literature.

  15. Gas-phase hydrogen/deuterium exchange of 5'- and 3'-mononucleotides in a quadrupole ion trap: exploring the role of conformation and system energy.

    Science.gov (United States)

    Chipuk, Joseph E; Brodbelt, Jennifer S

    2007-04-01

    Gas-phase hydrogen/deuterium (H/D) exchange reactions for deprotonated 2'-deoxy-5'-monophosphate and 2'-deoxy-3'-monophosphate nucleotides with D(2)O were performed in a quadrupole ion trap mass spectrometer. To augment these experiments, molecular modeling was also conducted to identify likely deprotonation sites and potential gas-phase conformations of the anions. A majority of the 5'-monophosphates exchanged extensively with several of the compounds completely incorporating deuterium in place of their labile hydrogen atoms. In contrast, most of the 3'-monophosphate isomers exchanged relatively few hydrogen atoms, even though the rate of the first two exchanges was greater than observed for the 5'-monophosphates. Mononucleotides that failed to incorporate more than two deuterium atoms under default reaction conditions were often found to exchange more extensively when reactions were performed under higher energy conditions. Integration of the experimental and theoretical results supports the use of a relay exchange mechanism and suggests that the exchange behavior depends highly on the identity and orientation of the nucleobase and the position and flexibility of the deprotonated phosphate moiety. These observations also highlight the importance of the distance between the various participating groups in addition to their gas-phase acidity and basicity.

  16. Synthesis, spectroscopy, and hydrogen/deuterium exchange in high-spin iron(II) hydride complexes.

    Science.gov (United States)

    Dugan, Thomas R; Bill, Eckhard; MacLeod, K Cory; Brennessel, William W; Holland, Patrick L

    2014-03-03

    Very few hydride complexes are known in which the metals have a high-spin electronic configuration. We describe the characterization of several high-spin iron(II) hydride/deuteride isotopologues and their exchange reactions with one another and with H2/D2. Though the hydride/deuteride signal is not observable in NMR spectra, the choice of isotope has an influence on the chemical shifts of distant protons in the dimers through the paramagnetic isotope effect on chemical shift. This provides the first way to monitor the exchange of H and D in the bridging positions of these hydride complexes. The rate of exchange depends on the size of the supporting ligand, and this is consistent with the idea that H2/D2 exchange into the hydrides occurs through the dimeric complexes rather than through a transient monomer. The understanding of H/D exchange mechanisms in these high-spin iron hydride complexes may be relevant to postulated nitrogenase mechanisms.

  17. Ion exchange in KTiOPO4 crystals irradiated by copper and hydrogen ions.

    Science.gov (United States)

    Zhang, Ruifeng; Lu, Fei; Lian, Jie; Liu, Hanping; Liu, Xiangzhi; Lu, Qingming; Ma, Hongji

    2008-05-12

    Cs(+)-K+ ion exchanges were produced on KTiOPO4 crystals which is prior irradiated by Cu+ can H+ ions. The energy and dose of implanted Cu+ ions are 1.5 MeV and 0.5 x 10(14) ions/cm2, and that of H+ are 300 keV and 1 x 10(16) ions/cm2, respectively. The temperature of ions exchange is 430 degrees C, and the time range from 15 minutes to 30 minutes. The prism coupling method is used to measure the dark mode spectra of the samples. Compared with results of ion exchange on the sample without irradiations, both the number of guided mode and its corresponding effective refractive index are decreased. The experimental results indicate that the ion exchange rate closely related with the lattice damage and the damage layers formed in the depth of maximum nuclear energy deposition act as a barrier to block the ions diffuse into the sample and the concentration of defects can modify the speed of ion exchange..

  18. Synthesis of amide-functionalized cellulose esters by olefin cross-metathesis.

    Science.gov (United States)

    Meng, Xiangtao; Edgar, Kevin J

    2015-11-05

    Cellulose esters with amide functionalities were synthesized by cross-metathesis (CM) reaction of terminally olefinic esters with different acrylamides, catalyzed by Hoveyda-Grubbs 2nd generation catalyst. Chelation by amides of the catalyst ruthenium center caused low conversions using conventional solvents. The effects of both solvent and structure of acrylamide on reaction conversion were investigated. While the inherent tendency of acrylamides to chelate Ru is governed by the acrylamide N-substituents, employing acetic acid as a solvent significantly improved the conversion of certain acrylamides, from 50% to up to 99%. Homogeneous hydrogenation using p-toluenesulfonyl hydrazide successfully eliminated the α,β-unsaturation of the CM products to give stable amide-functionalized cellulose esters. The amide-functionalized product showed higher Tg than its starting terminally olefinic counterpart, which may have resulted from strong hydrogen bonding interactions of the amide functional groups. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Protein-like proton exchange in a synthetic host cavity.

    Science.gov (United States)

    Hart-Cooper, William M; Sgarlata, Carmelo; Perrin, Charles L; Toste, F Dean; Bergman, Robert G; Raymond, Kenneth N

    2015-12-15

    The mechanism of proton exchange in a metal-ligand enzyme active site mimic (compound 1) is described through amide hydrogen-deuterium exchange kinetics. The type and ratio of cationic guest to host in solution affect the rate of isotope exchange, suggesting that the rate of exchange is driven by a host whose cavity is occupied by water. Rate constants for acid-, base-, and water-mediated proton exchange vary by orders of magnitude depending on the guest, and differ by up to 200 million-fold relative to an alanine polypeptide. These results suggest that the unusual microenvironment of the cavity of 1 can dramatically alter the reactivity of associated water by magnitudes comparable to that of enzymes.

  20. Catalytic activity of superconducting ceramics of Y-Ba-Cu-O type in reaction of H-D exchange of molecular hydrogen

    International Nuclear Information System (INIS)

    Parbuzin, V.S.; Gul'yants, V.V.

    1989-01-01

    Catalytic activity of high-temperature superconducting oxide ceramics of Y-Ba-Cu-O type in reaction of deuterium-hydrogen exchange was investigated under chromatographic conditions. Rate constants of the reaction and activation energy of the process, equal to 58 ± 2 kJ/mol, were determined in 350 - 450 K range and at 18250Pa hydrogen pressure. Assumption about applicability of Bonhoeffer-Farkas mechanism with adsorption on copper atoms, adjoinig oxygen vacancies, was made

  1. Hydrogen.

    Science.gov (United States)

    Bockris, John O'M

    2011-11-30

    The idea of a "Hydrogen Economy" is that carbon containing fuels should be replaced by hydrogen, thus eliminating air pollution and growth of CO₂ in the atmosphere. However, storage of a gas, its transport and reconversion to electricity doubles the cost of H₂ from the electrolyzer. Methanol made with CO₂ from the atmosphere is a zero carbon fuel created from inexhaustible components from the atmosphere. Extensive work on the splitting of water by bacteria shows that if wastes are used as the origin of feed for certain bacteria, the cost for hydrogen becomes lower than any yet known. The first creation of hydrogen and electricity from light was carried out in 1976 by Ohashi et al. at Flinders University in Australia. Improvements in knowledge of the structure of the semiconductor-solution system used in a solar breakdown of water has led to the discovery of surface states which take part in giving rise to hydrogen (Khan). Photoelectrocatalysis made a ten times increase in the efficiency of the photo production of hydrogen from water. The use of two electrode cells; p and n semiconductors respectively, was first introduced by Uosaki in 1978. Most photoanodes decompose during the photoelectrolysis. To avoid this, it has been necessary to create a transparent shield between the semiconductor and its electronic properties and the solution. In this way, 8.5% at 25 °C and 9.5% at 50 °C has been reached in the photo dissociation of water (GaP and InAs) by Kainthla and Barbara Zeleney in 1989. A large consortium has been funded by the US government at the California Institute of Technology under the direction of Nathan Lewis. The decomposition of water by light is the main aim of this group. Whether light will be the origin of the post fossil fuel supply of energy may be questionable, but the maximum program in this direction is likely to come from Cal. Tech.

  2. Mixed filling for the successive isotopic exchange in the phase sequence water - water vapors - hydrogen

    International Nuclear Information System (INIS)

    Stefanescu, D.; Peculea, M.; Hirean, I.; Croitoru, C.

    1995-01-01

    The paper deals with the process of the isotopic exchange implied in heavy water production. Details concerning the structural arrangement of the process contact elements inside the exchange columns are presented. A hydrophilic filling, based on phosphorous bronze, and the platinum catalyst structure , resulted from this work, are to be implemented in the column equipment of the heavy water distillation pilot operating in connection with the CANDU type reactors. The performances of the mixed catalyst components were derived from experimental data by means of the three fluids model equations

  3. Phase space investigation of the lithium amide halides

    Energy Technology Data Exchange (ETDEWEB)

    Davies, Rosalind A. [Hydrogen Storage Chemistry Group, School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (United Kingdom); Hydrogen and Fuel Cell Group, School of Chemical Engineering, University of Birmingham, Edgbaston B15 2TT (United Kingdom); Hewett, David R.; Korkiakoski, Emma [Hydrogen Storage Chemistry Group, School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (United Kingdom); Thompson, Stephen P. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0QX (United Kingdom); Anderson, Paul A., E-mail: p.a.anderson@bham.ac.uk [Hydrogen Storage Chemistry Group, School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (United Kingdom)

    2015-10-05

    Highlights: • The lower limits of halide incorporation in lithium amide have been investigated. • The only amide iodide stoichiometry observed was Li{sub 3}(NH{sub 2}){sub 2}I. • Solid solutions were observed in both the amide chloride and amide bromide systems. • A 46% reduction in chloride content resulted in a new phase: Li{sub 7}(NH{sub 2}){sub 6}Cl. • New low-chloride phase maintained improved H{sub 2} desorption properties of Li{sub 4}(NH{sub 2}){sub 3}Cl. - Abstract: An investigation has been carried out into the lower limits of halide incorporation in lithium amide (LiNH{sub 2}). It was found that the lithium amide iodide Li{sub 3}(NH{sub 2}){sub 2}I was unable to accommodate any variation in stoichiometry. In contrast, some variation in stoichiometry was accommodated in Li{sub 7}(NH{sub 2}){sub 6}Br, as shown by a decrease in unit cell volume when the bromide content was reduced. The amide chloride Li{sub 4}(NH{sub 2}){sub 3}Cl was found to adopt either a rhombohedral or a cubic structure depending on the reaction conditions. Reduction in chloride content generally resulted in a mixture of phases, but a new rhombohedral phase with the stoichiometry Li{sub 7}(NH{sub 2}){sub 6}Cl was observed. In comparison to LiNH{sub 2}, this new low-chloride phase exhibited similar improved hydrogen desorption properties as Li{sub 4}(NH{sub 2}){sub 3}Cl but with a much reduced weight penalty through addition of chloride. Attempts to dope lithium amide with fluoride ions have so far proved unsuccessful.

  4. Separation of tautomeric forms of [2-nitrophloroglucinol-H]- by an in-electrospray ionization source hydrogen/deuterium exchange approach.

    Science.gov (United States)

    Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Starodubtseva, Natalia; Kukaev, Eugene; Nikotaev, Eugene

    2014-01-01

    Here we report the observation that, depending on the solvent used for the electrospray, 2-nitrophloroglucinol undergoes a deprotona- tion from different sites forming two tautomeric gas phase ions. Those ions differ bythe collision-induced dissociation [CID] spectra and by the gas phase hydrogen/deuterium (H/D) exchange kinetic. We performed H/D exchange in the electrospray ionization (ESI) source by saturation ESI region with vapors of deuterated solvent (D20). It was observed that [2-nitrophloroglucinol-H]- exchanges two -OH hydrogens when MeOD is used as the spray solvent but when the spray solvent is 50:50 MeOD/DO20 we observed an additional two H/D exchanges at the aromatic ring. We propose that the reaction occurs via a keto-enolt tautomerization mechanism which was found to be energetically favorable.

  5. Amides in Nature and Biocatalysis

    NARCIS (Netherlands)

    Pitzer, J.; Steiner, K.

    2016-01-01

    Amides are widespread in biologically active compounds with a broad range of applications in biotechnology, agriculture and medicine. Therefore, as alternative to chemical synthesis the biocatalytic amide synthesis is a very interesting field of research. As usual, Nature can serve as guide in the

  6. QUDeX-MS: hydrogen/deuterium exchange calculation for mass spectra with resolved isotopic fine structure.

    Science.gov (United States)

    Salisbury, Joseph P; Liu, Qian; Agar, Jeffrey N

    2014-12-11

    Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation. Here, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com . Isotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX

  7. Phosphoinositide binding regulates alpha-actinin CH2 domain structure: analysis by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Full, Stephen J; Deinzer, Max L; Ho, P Shing; Greenwood, Jeffrey A

    2007-12-01

    alpha-Actinin is an actin bundling protein that regulates cell adhesion by directly linking actin filaments to integrin adhesion receptors. Phosphatidylinositol (4,5)-diphosphate (PtdIns (4,5)-P(2)) and phosphatidylinositol (3,4,5)-triphosphate (PtdIns (3,4,5)-P(3)) bind to the calponin homology 2 domain of alpha-actinin, regulating its interactions with actin filaments and integrin receptors. In this study, we examine the mechanism by which phosphoinositide binding regulates alpha-actinin function using mass spectrometry to monitor hydrogen-deuterium (H/D) exchange within the calponin homology 2 domain. The overall level of H/D exchange for the entire protein showed that PtdIns (3,4,5)-P(3) binding alters the structure of the calponin homology 2 domain increasing deuterium incorporation, whereas PtdIns (4,5)-P(2) induces changes in the structure decreasing deuterium incorporation. Analysis of peptic fragments from the calponin homology 2 domain showed decreased local H/D exchange within the loop region preceding helix F with both phosphoinositides. However, the binding of PtdIns (3,4,5)-P(3) also induced increased exchange within helix E. This suggests that the phosphate groups on the fourth and fifth position of the inositol head group of the phosphoinositides constrict the calponin homology 2 domain, thereby altering the orientation of actin binding sequence 3 and decreasing the affinity of alpha-actinin for filamentous actin. In contrast, the phosphate group on the third position of the inositol head group of PtdIns (3,4,5)-P(3) perturbs the calponin homology 2 domain, altering the interaction between the N and C terminus of the full-length alpha-actinin antiparallel homodimer, thereby disrupting bundling activity and interaction with integrin receptors.

  8. Preparation by ion exchange and structural simulation of a new hydrogen phosphate of sodium zirconium

    International Nuclear Information System (INIS)

    Contreras R, A.; Fernandez V, S. M.; Ordonez R, E.; Perez A, M.

    2008-01-01

    It is described the method of synthesis of the τ-Zr P and the obtaining of its sodium form by ion exchange, the simulation of crystalline model and their patterns of X-ray diffraction and comparison of these with other compounds reported in the literature. (Author)

  9. On the hydroxylic hydrogen isotopic exchange for infrared studies of hydrated solids

    International Nuclear Information System (INIS)

    Versaud, P.-C.; Lenoir, Jeanine

    1979-01-01

    From the constant of the equilibrium H 2 O + D 2 O reversible 2HOD and the deuterium content of heavy water, we can show that a single technique allows one to reach the limit of the isotopic exchange at the ambient temperature. This technique consists necessarily of successive introductions of D 2 O vapour followed every time by adequate vacuum [fr

  10. Hydrogen/Denterium exchange during n.butane conversion on H-ZSM-5

    NARCIS (Netherlands)

    Narbeshuber, T.; Narbeshuber, Thomas F.; Stockenhuber, Michael; Brait, Axel; Brait, A.; Seshan, Kulathuiyer; Lercher, J.A.

    1996-01-01

    Steady-state isotope tracer studies and isotope transient response experiments ofn-butane conversion on H-ZSM-5 (Si/Al = 35) were carried out between 673 and 823 K. Among the three main reactions, the rate of H/D-exchange is at least one order of magnitude higher compared to the rates of cracking or

  11. Photochemical hydrogen-deuterium exchange reaction of tryptophan: the role in nonradiative decay of singlet tryptophan

    International Nuclear Information System (INIS)

    Saito, I.; Sugiyama, H.; Yamamoto, A.; Muramatsu, S.; Matsuura, T.

    1984-01-01

    The mechanism of nonradiative decay of singlet excited tryptophan (Trp) in aqueous solution was investigated by a highly selective photosubstitution of the C-4 hydrogen of Trp with deuterium of solvent D 2 O. It was concluded that intramolecular proton transfer from the α-ammonia group giving rise to formation of a protonated species plays an important role in the nonradiative decay of singlet Trp at neutral pH. 11 references, 1 figure

  12. KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jia; Harrison, Rane A.; Li, Lianbo; Zeng, Mei; Gondi, Sudershan; Scott, David; Gray, Nathanael S.; Engen, John R.; Westover, Kenneth D. (NEU); (DFCI); (UTSMC); (Harvard-Med)

    2017-09-01

    KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basis for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.

  13. Activity-regulating structural changes and autoantibody epitopes in transglutaminase 2 assessed by hydrogen/deuterium exchange

    DEFF Research Database (Denmark)

    Iversen, Rasmus; Mysling, Simon; Hnida, Kathrin

    2014-01-01

    The multifunctional enzyme transglutaminase 2 (TG2) is the target of autoantibodies in the gluten-sensitive enteropathy celiac disease. In addition, the enzyme is responsible for deamidation of gluten peptides, which are subsequently targeted by T cells. To understand the regulation of TG2 activity...... and the enzyme's role as an autoantigen in celiac disease, we have addressed structural properties of TG2 in solution by using hydrogen/deuterium exchange monitored by mass spectrometry. We demonstrate that Ca(2+) binding, which is necessary for TG2 activity, induces structural changes in the catalytic core...... domain of the enzyme. Cysteine oxidation was found to abolish these changes, suggesting a mechanism whereby disulfide bond formation inactivates the enzyme. Further, by using TG2-specific human monoclonal antibodies generated from intestinal plasma cells of celiac disease patients, we observed...

  14. Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry.

    Science.gov (United States)

    Resetca, Diana; Wilson, Derek J

    2013-11-01

    This review outlines the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) and hydrogen-deuterium exchange (HDX) to study rapid, activity-linked conformational transitions in proteins. The method is implemented on a microfluidic chip which incorporates all sample-handling steps required for a 'bottom-up' HDX workflow: a capillary mixer for sub-second HDX labeling, a static mixer for HDX quenching, a microreactor for rapid protein digestion, and on-chip electrospray. By combining short HDX labeling pulses with rapid digestion, this approach provides a detailed characterization of the structural transitions that occur during protein folding, ligand binding, post-translational modification and catalytic turnover in enzymes. This broad spectrum of applications in areas largely inaccessible to conventional techniques means that microfluidics-enabled TRESI-MS/HDX is a unique and powerful approach for investigating the dynamic basis of protein function. © 2013 FEBS.

  15. Conformational changes in the g protein-coupled receptor rhodopsin revealed by histidine hydrogen-deuterium exchange.

    Science.gov (United States)

    Lodowski, David T; Palczewski, Krzysztof; Miyagi, Masaru

    2010-11-09

    G protein-coupled receptors (GPCRs) are activated by ligand binding, allowing extracellular signals to be efficiently transmitted through the membrane to the G protein recognition site, 40 Å away. Utilizing His residues found spaced throughout the GPCR, rhodopsin, we used His hydrogen-deuterium exchange (His-HDX) to monitor long-time scale structural rearrangements previously inaccessible by other means. The half-lives of His-HDX indicate clear differences in the solvent accessibility of three His residues in rhodopsin/opsin and Zn2+-dependent changes in the pKa for His195. These results indicate the utility of His-HDX in examining structural rearrangements in native source and membrane proteins without requiring structural modification.

  16. Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution.

    Science.gov (United States)

    Zhu, Shaolong; Campbell, J Larry; Chernushevich, Igor; Le Blanc, J C Yves; Wilson, Derek J

    2016-06-01

    Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (DMS-field asymmetric waveform ion mobility spectroscopy (FAIMS)-the application of DMS to intact biomacromolecules remains largely unexplored. In this work, we employ DMS combined with gas-phase hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development. Graphical Abstract ᅟ.

  17. Iridium(I)-catalyzed regioselective C-H activation and hydrogen-isotope exchange of non-aromatic unsaturated functionality.

    Science.gov (United States)

    Kerr, William J; Mudd, Richard J; Paterson, Laura C; Brown, Jack A

    2014-11-03

    Isotopic labelling is a key technology of increasing importance for the investigation of new CH activation and functionalization techniques, as well as in the construction of labelled molecules for use within both organic synthesis and drug discovery. Herein, we report for the first time selective iridium-catalyzed CH activation and hydrogen-isotope exchange at the β-position of unsaturated organic compounds. The use of our highly active [Ir(cod)(IMes)(PPh3 )][PF6 ] (cod=1,5-cyclooctadiene) catalyst, under mild reaction conditions, allows the regioselective β-activation and labelling of a range of α,β-unsaturated compounds with differing steric and electronic properties. This new process delivers high levels of isotope incorporation over short reaction times by using low levels of catalyst loading. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Mapping the H-NOX/HK Binding Interface in Vibrio cholerae by Hydrogen/Deuterium Exchange Mass Spectrometry.

    Science.gov (United States)

    Guo, Yirui; Iavarone, Anthony T; Cooper, Matthew M; Marletta, Michael A

    2018-03-20

    Heme-nitric oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O 2 ). H-NOX proteins typically regulate histidine kinases (HK) located within the same operon. It has been reported that NO-bound H-NOXs inhibit cognate histidine kinase autophosphorylation in bacterial H-NOX/HK complexes; however, a detailed mechanism of NO-mediated regulation of the H-NOX/HK activity remains unknown. In this study, the binding interface of Vibrio cholerae ( Vc) H-NOX/HK complex was characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and further validated by mutagenesis, leading to a new model for NO-dependent kinase inhibition. A conformational change in Vc H-NOX introduced by NO generates a new kinase-binding interface, thus locking the kinase in an inhibitory conformation.

  19. Mg2+ dependence of 70 S ribosomal protein flexibility revealed by hydrogen/deuterium exchange and mass spectrometry.

    Science.gov (United States)

    Yamamoto, Tatsuya; Shimizu, Yoshihiro; Ueda, Takuya; Shiro, Yoshitsugu

    2010-02-19

    The ribosome from Escherichia coli requires a specific concentration of Mg(2+) to maintain the 70 S complex formation and allow protein synthesis, and then the structure must be stable and flexible. How does the ribosome acquire these conflicting factors at the same time? Here, we investigated the hydrogen/deuterium exchange of 52 proteins in the 70 S ribosome, which controlled stability and flexibility under various Mg(2+) concentrations, using mass spectrometry. Many proteins exhibited a sigmoidal curve for Mg(2+) concentration dependence, incorporating more deuterium at lower Mg(2+) concentration. By comparing deuterium incorporation with assembly, we have discovered a typical mechanism of complexes for acquiring both stability and flexibility at the same time. In addition, we got information of the localization of flexibility in ribosomal function by the analysis of related proteins with stalk protein, tRNA, mRNA, and nascent peptide, and demonstrate the relationship between structure, assembly, flexibility, and function of the ribosome.

  20. Deuterium exchange reaction between hydrogen and water in a trickle-bed column packed with novel catalysts

    International Nuclear Information System (INIS)

    Ahn, D. H.; Baek, S. W.; Lee, H. S.; Kim, K. R.; Kang, H. S.; Lee, S. H.; Jeong, H. S.

    1998-01-01

    The activity of a novel catalyst (Pt/SDBC) for deuterium exchange reaction between water and hydrogen streams in a trickle bed was measured. The performance of the catalyst was compared with a commercial catalyst with same metal content. The catalytic activity for the bed of wet-proofed catalyst diluted with hydrophillic packing material also measured. The Pt/SDBC catalyst shows higher activity in the liquid phase reaction than the commercial catalyst as measured in the vapor phase reaction. The performance for 50% dilution of the Pt/SDBC catalyst bed with hydrophillic packing material is better than that of the 100% bed due to more liquid holdup and better water distribution

  1. Hydrogen

    Directory of Open Access Journals (Sweden)

    John O’M. Bockris

    2011-11-01

    Full Text Available The idea of a “Hydrogen Economy” is that carbon containing fuels should be replaced by hydrogen, thus eliminating air pollution and growth of CO2 in the atmosphere. However, storage of a gas, its transport and reconversion to electricity doubles the cost of H2 from the electrolyzer. Methanol made with CO2 from the atmosphere is a zero carbon fuel created from inexhaustible components from the atmosphere. Extensive work on the splitting of water by bacteria shows that if wastes are used as the origin of feed for certain bacteria, the cost for hydrogen becomes lower than any yet known. The first creation of hydrogen and electricity from light was carried out in 1976 by Ohashi et al. at Flinders University in Australia. Improvements in knowledge of the structure of the semiconductor-solution system used in a solar breakdown of water has led to the discovery of surface states which take part in giving rise to hydrogen (Khan. Photoelectrocatalysis made a ten times increase in the efficiency of the photo production of hydrogen from water. The use of two electrode cells; p and n semiconductors respectively, was first introduced by Uosaki in 1978. Most photoanodes decompose during the photoelectrolysis. To avoid this, it has been necessary to create a transparent shield between the semiconductor and its electronic properties and the solution. In this way, 8.5% at 25 °C and 9.5% at 50 °C has been reached in the photo dissociation of water (GaP and InAs by Kainthla and Barbara Zeleney in 1989. A large consortium has been funded by the US government at the California Institute of Technology under the direction of Nathan Lewis. The decomposition of water by light is the main aim of this group. Whether light will be the origin of the post fossil fuel supply of energy may be questionable, but the maximum program in this direction is likely to come from Cal. Tech.

  2. Oligonucleotide gas-phase hydrogen/deuterium exchange with D2S in the collision cell of a quadrupole-Fourier transform ion cyclotron resonance mass spectrometer.

    Science.gov (United States)

    Mo, Jingjie; Håkansson, Kristina

    2007-10-15

    We have implemented gas-phase hydrogen/deuterium exchange (HDX) experiments in the external collision cell of a hybrid quadrupole-Fourier transform ion cyclotron resonance mass spectrometer. In this configuration, multiply charged oligonucleotide anions undergo significant exchange with D(2)S at reaction intervals ranging from 0.11 to 60.1 s. For DNA homohexamers, relative exchange rates were dC(6) approximately dA(6) > dG(6) > dT(6), correlating with the gas-phase acidities of nucleobases (C > A > T > G), except for guanine. Our results are consistent with a relay mechanism in which D(2)S interacts with both a backbone phosphate group and a neutral nucleobase through hydrogen bonding. We propose that the faster exchange of polyguanosine compared to polythymidine is due to the larger size of guanine and the orientation of its labile hydrogens, which may result in gas-phase conformations more favorable for forming complexes with D(2)S. Similar trends were observed for RNA homohexamers, although their HDX rates were faster than for DNA, suggesting they can also exchange via another relay process involving the 2'-hydroxyl group. HDX of DNA duplexes further supports the involvement of nucleobase hydrogens because duplexes exchanged slower than their corresponding single strands, presumably due to the intermolecular hydrogen bonds between nucleobases. This work constitutes the first investigation of the mechanisms of oligonucleotide gas-phase HDX. Our results on duplexes show promise for application of this strategy to the characterization of structured nucleic acids.

  3. Deuterium retention in tungsten after heavy ion damage and hydrogen isotope exchange in PISCES

    Science.gov (United States)

    Barton, J. L.; Wang, Y. Q.; Dittmar, T.; Doerner, R. P.; Tynan, G. R.

    2014-08-01

    The effect of H isotope exchange and radiation damage on the retention of D in W was examined in the PISCES linear plasma device. W samples were treated with D plasma at low sample temperatures (473 K), with a fluence of 1026 ions/m2 and ion energies of 150 eV. Each sample was then exposed to varying doses of H plasma with similar sample temperature and plasma conditions to fluences ranging from 0 to 1026 ions/m2, to examine the effectiveness of isotope exchange as a means of tritium removal. The D(3He, p)4He nuclear reaction was used to measure D concentration profiles up to a depth of 7.7 μm. Thermal desorption spectroscopy (TDS) was used to determine the D retained throughout the bulk of the sample. Isotope exchange allows for a unique study of atomic migration by separately examining the diffusion of implanted atoms from those bombarding the surface. D atoms are exchanged out of traps as a result of H plasma bombardment and diffuse until either falling into another trap or reaching the surface to recombine and escape. Radiation damage at levels of 0.01, 0.1, and 1 displacements per atom (dpa) was carried out before plasma exposure on some samples with 2 MeV Cu ions as a surrogate for damage caused by fusion neutrons. The Cu ion damage was compared to damage induced by 6 MeV W ions to see if there is an effect of Cu contamination on retention. We saw little difference in Cu versus W ion damage at low dpa, but at 1 dpa, where Cu content reached 65 appm, contamination seems to be significant. Retention measurements showed that ion damage has little effectiveness on isotope removal at these sample temperatures; however, there is evidence to suggest that the trapping mechanisms in W change as damage is increased.

  4. Simultaneous Blood–Tissue Exchange of Oxygen, Carbon Dioxide, Bicarbonate, and Hydrogen Ion

    Science.gov (United States)

    Dash, Ranjan K.; Bassingthwaighte, James B.

    2014-01-01

    A detailed nonlinear four-region (red blood cell, plasma, interstitial fluid, and parenchymal cell) axially distributed convection-diffusion-permeation-reaction-binding computational model is developed to study the simultaneous transport and exchange of oxygen (O2) and carbon dioxide (CO2) in the blood–tissue exchange system of the heart. Since the pH variation in blood and tissue influences the transport and exchange of O2 and CO2 (Bohr and Haldane effects), and since most CO2 is transported as HCO3- (bicarbonate) via the CO2 hydration (buffering) reaction, the transport and exchange of HCO3- and H+ are also simulated along with that of O2 and CO2. Furthermore, the model accounts for the competitive nonlinear binding of O2 and CO2 with the hemoglobin inside the red blood cells (nonlinear O2–CO2 interactions, Bohr and Haldane effects), and myoglobin-facilitated transport of O2 inside the parenchymal cells. The consumption of O2 through cytochrome-c oxidase reaction inside the parenchymal cells is based on Michaelis–Menten kinetics. The corresponding production of CO2 is determined by respiratory quotient (RQ), depending on the relative consumption of carbohydrate, protein, and fat. The model gives a physiologically realistic description of O2 transport and metabolism in the microcirculation of the heart. Furthermore, because model solutions for tracer transients and steady states can be computed highly efficiently, this model may be the preferred vehicle for routine data analysis where repetitive solutions and parameter optimization are required, as is the case in PET imaging for estimating myocardial O2 consumption. PMID:16775761

  5. Modeling the performance of hydrogen-oxygen unitized regenerative proton exchange membrane fuel cells for energy storage

    Science.gov (United States)

    Guarnieri, Massimo; Alotto, Piergiorgio; Moro, Federico

    2015-11-01

    Thanks to the independent sizing of power and energy, hydrogen-based energy storage is one of the very few technologies capable of providing long operational times in addition to the other advantages offered by electrochemical energy storage, for example scalability, site versatility, and mobile service. The typical design consists of an electrolyzer in charge mode and a separate fuel cell in discharge mode. Instead, a unitized regenerative fuel cell (URFC) is a single device performing both energy conversions, achieving a higher compactness and power-to-weight ratio. This paper presents a performance model of a URFC based on a proton exchange membrane (PEM) electrolyte and working on hydrogen and oxygen, which can provide high energy and power densities (>0.7 W cm-2). It provides voltage, power, and efficiency at varying load conditions as functions of the controlling physical quantities: temperature, pressure, concentration, and humidification. The model constitutes a tool for designing the interface and control sub-system as well as for exploring optimized cell/stack designs and operational conditions. To date, only a few of such analyses have been carried out and more research is needed in order to explore the true potential of URFCs.

  6. Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 3. Estimating Surface Area Exposure by Deuterium Uptake

    Science.gov (United States)

    Khakinejad, Mahdiar; Ghassabi Kondalaji, Samaneh; Donohoe, Gregory C.; Valentine, Stephen J.

    2016-03-01

    Gas-phase hydrogen deuterium exchange (HDX), collision cross section (CCS) measurement, and molecular dynamics simulation (MDS) techniques were utilized to develop and compare three methods for estimating the relative surface area exposure of separate peptide chains within bovine insulin ions. Electrosprayed [M - 3H]3- and [M - 5H]5- insulin ions produced a single conformer type with respective collision cross sections of 528 ± 5 Å2 and 808 ± 2 Å2. [M - 4H]4- ions were comprised of more compact (Ω = 676 ± 3 Å2) and diffuse (i.e., more elongated, Ω = 779 ± 3 Å2) ion conformer types. Ions were subjected to HDX in the drift tube using D2O as the reagent gas. Collision-induced dissociation was used to fragment mobility-selected, isotopically labeled [M - 4H]4- and [M - 5H]5- ions into the protein subchains. Deuterium uptake levels of each chain can be explained by limited inter-chain isotopic scrambling upon collisional activation. Using nominal ion structures from MDS and a hydrogen accessibility model, the deuterium uptake for each chain was correlated to its exposed surface area. In separate experiments, the per-residue deuterium content for the protonated and deprotonated ions of the synthetic peptide KKDDDDDIIKIIK were compared. The differences in deuterium content indicated the regional HDX accessibility for cations versus anions. Using ions of similar conformational type, this comparison highlights the complementary nature of HDX data obtained from positive- and negative-ion analysis.

  7. Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics.

    Science.gov (United States)

    Lento, Cristina; Zhu, Shaolong; Brown, Kerene A; Knox, Ruth; Liuni, Peter; Wilson, Derek J

    2017-04-17

    Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

  8. Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 2. Assessing Charge Site Location and Isotope Scrambling

    Science.gov (United States)

    Khakinejad, Mahdiar; Ghassabi Kondalaji, Samaneh; Donohoe, Gregory C.; Valentine, Stephen J.

    2016-03-01

    Ion mobility spectrometry (IMS) coupled with gas-phase hydrogen deuterium exchange (HDX)-mass spectrometry (MS) and molecular dynamic simulations (MDS) has been used for structural investigation of anions produced by electrospraying a sample containing a synthetic peptide having the sequence KKDDDDDIIKIIK. In these experiments the potential of the analytical method for locating charge sites on ions as well as for utilizing collision-induced dissociation (CID) to reveal the degree of deuterium uptake within specific amino acid residues has been assessed. For diffuse (i.e., more elongated) [M - 2H]2- ions, decreased deuterium content along with MDS data suggest that the D4 and D6 residues are charge sites, whereas for the more diffuse [M - 3H]3- ions, the data suggest that the D4, D7, and the C-terminus are deprotonated. Fragmentation of mobility-selected, diffuse [M - 2H]2- ions to determine deuterium uptake at individual amino acid residues reveals a degree of deuterium retention at incorporation sites. Although the diffuse [M - 3H]3- ions may show more HD scrambling, it is not possible to clearly distinguish HD scrambling from the expected deuterium uptake based on a hydrogen accessibility model. The capability of the IMS-HDX-MS/MS approach to provide relevant details about ion structure is discussed. Additionally, the ability to extend the approach for locating protonation sites on positively-charged ions is presented.

  9. Ion Mobility Spectrometry-Mass Spectrometry Coupled with Gas-Phase Hydrogen/Deuterium Exchange for Metabolomics Analyses

    Science.gov (United States)

    Maleki, Hossein; Karanji, Ahmad K.; Majuta, Sandra; Maurer, Megan M.; Valentine, Stephen J.

    2018-02-01

    Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate ( in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract.

  10. Hydrogen isotope trapping on graphite collectors during an isotope exchange experiment in the Tokamak Fusion Test Reactor

    International Nuclear Information System (INIS)

    Kilpatrick, S.J.; Ulrickson, M.; Dylla, H.F.; Manos, D.M.; Ramsey, A.T.; Nygren, R.; Hirooka, Y.; Wampler, W.R.

    1989-03-01

    A rotatable collector probe was used to expose several graphite samples to a deuterium-to-hydrogen-to-deuterium exchange experiment in the Tokamak Fusion Test Reactor (TFTR) at the start of the 1988 operations period. This experiment proved the utility of helium conditioning discharges in accelerating the changeover process. Samples included portions of a tile taken from the inner bumper limiter (POCO AXF-5Q graphite) of TFTR during the recent machine opening, and coupons which had been conditioned in the Plasma Interactive Surface Component Experimental Station (PISCES) by exposure to a helium plasma. The samples were exposed to different groups of the /approximately/100 1.4 MA discharges that comprised the experiment. They were removed and analyzed for retained deuterium and impurities by nuclear reaction analysis and Rutherford backscattering spectrometry. Codeposited carbon layers had been formed with thicknesses up to several tenths of a micron. The inferred percentages of trapped hydrogenic species were in general agreement with spectroscopic data. The computed carbon fluence per D + discharge, 1.2 /times/ 10 17 C/cm 2 , is compared to recent measurements on limiter tiles removed from TFTR. 22 refs., 3 figs., 1 tab

  11. Hydrogen isotope trapping on graphite collectors during an isotope exchange experiment in the tokomak fusion test reactor

    International Nuclear Information System (INIS)

    Kilpatrick, S.J.; Nygren, R.; Wampler, W.R.; Ulrickson, M.; Dylla, H.F.; Manos, D.M.; Ramsey, A.T.; Hirooka, Y.

    1988-01-01

    A rotatable collector probe was used to expose several graphite samples to a deuterium-to-hydrogen-to-deuterium exchange experiment in the Tokamak Fusion Test Reactor (TFTR) at the start of the 1988 operations period. This experiment proved the utility of helium conditioning discharges in accelerating the changeover process. Samples included portions of a tile taken from the inner bumper limiter (POCO AXF-5Q graphite) of TFTR during the recent machine opening, and coupons which had been conditioned in the Plasma Surface Interaction Experimental Facility (PISCES) by exposure to a helium plasma. The samples were exposed to different groups of the /approximately/100 1.4MA discharges that comprised the experiment. They were removed and analyzed for retained deuterium and impurities by nuclear reaction analysis and Rutherford backscattering spectrometry. Codeposited carbon layers had been formed with thicknesses up to several tenths of a micron. The inferred percentages of trapped hydrogenic species were in general agreement with spectroscopic data. The computed carbon fluence per D + discharge, 1.2 /times/ 10 17 C/cm 2 , is compared to recent measurements on limiter tiles removed from TFTR. 21 refs., 3 figs., 1 tab

  12. Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

    Directory of Open Access Journals (Sweden)

    Vogtt K.

    2005-01-01

    Full Text Available COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC and under high pressure conditions at low temperature (3.75 kbar, -13ºC. Moreover, the influence of co-solvents (sorbitol, urea on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

  13. The dissolution of organic ion exchange resins using iron-catalysed hydrogen peroxide

    International Nuclear Information System (INIS)

    Hawkings, N.; Horton, K.D.; Snelling, K.W.

    1980-10-01

    Feasibility studies have been made of the dissolution/partial decomposition of radioactive waste resins by means of iron-catalysed hydrogen peroxide. They have shown that the procedure is limited in its application and successfully treats only polystyrene/divinylbenzene-based resins. Evaporation of the final solution produces a solid residue which is difficult to handle and results in only a relatively small reduction in volume. It is concluded that the method could be used to dissolve specific waste resins for easier handling and disposal, but is not of general applicability. (author)

  14. Variable-temperature Fourier transform near-infrared imaging spectroscopy of the deuterium/hydrogen exchange in liquid D₂O.

    Science.gov (United States)

    Unger, Miriam; Ozaki, Yukihiro; Siesler, Heinz W

    2014-01-01

    In the present publication, the deuterium/hydrogen (D/H) exchange of liquid D2O exposed to water vapor of the surrounding atmosphere has been studied by variable-temperature Fourier transform near-infrared (FT-NIR) imaging spectroscopy. Apart from the visualization of the exchange process in the time-resolved FT-NIR images, kinetic parameters and the activation energy for this D/H exchange reaction have been derived from the Arrhenius plot of the variable-temperature spectroscopic data.

  15. Supercritical fluid chromatography coupled with in-source atmospheric pressure ionization hydrogen/deuterium exchange mass spectrometry for compound speciation.

    Science.gov (United States)

    Cho, Yunju; Choi, Man-Ho; Kim, Byungjoo; Kim, Sunghwan

    2016-04-29

    An experimental setup for the speciation of compounds by hydrogen/deuterium exchange (HDX) with atmospheric pressure ionization while performing chromatographic separation is presented. The proposed experimental setup combines the high performance supercritical fluid chromatography (SFC) system that can be readily used as an inlet for mass spectrometry (MS) and atmospheric pressure photo ionization (APPI) or atmospheric pressure chemical ionization (APCI) HDX. This combination overcomes the limitation of an approach using conventional liquid chromatography (LC) by minimizing the amount of deuterium solvents used for separation. In the SFC separation, supercritical CO2 was used as a major component of the mobile phase, and methanol was used as a minor co-solvent. By using deuterated methanol (CH3OD), AP HDX was achieved during SFC separation. To prove the concept, thirty one nitrogen- and/or oxygen-containing standard compounds were analyzed by SFC-AP HDX MS. The compounds were successfully speciated from the obtained SFC-MS spectra. The exchange ions were observed with as low as 1% of CH3OD in the mobile phase, and separation could be performed within approximately 20min using approximately 0.24 mL of CH3OD. The results showed that SFC separation and APPI/APCI HDX could be successfully performed using the suggested method. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Effects of sucrose and benzyl alcohol on GCSF conformational dynamics revealed by hydrogen deuterium exchange mass spectrometry.

    Science.gov (United States)

    Zhang, Jun; Banks, Douglas D; He, Feng; Treuheit, Michael J; Becker, Gerald W

    2015-05-01

    Protein stability, one of the major concerns for therapeutic protein development, can be optimized during process development by evaluating multiple formulation conditions. This can be a costly and lengthy procedure where different excipients and storage conditions are tested for their impact on protein stability. A better understanding of the effects of different formulation conditions at the molecular level will provide information on the local interactions within the protein leading to a more rational design of stable and efficacious formulations. In this study, we examined the roles of the excipients, sucrose and benzyl alcohol, on the conformational dynamics of recombinant human granulocyte colony stimulating factor using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS). Under physiological pH and temperature, sucrose globally protects the whole molecule from deuterium uptake, whereas benzyl alcohol induces increased deuterium uptake of the regions within the α-helical bundle, with even larger extent. The HDX experiments described were incorporated a set of internal peptides (Zhang et al., 2012. Anal Chem 84:4942-4949) to monitor the differences in intrinsic exchange rates in different formulations. In addition, we discussed the feasibility of implementing HDX-MS with these peptide probes in protein formulation development. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  17. Gas-phase hydrogen/deuterium exchange in a traveling wave ion guide for the examination of protein conformations.

    Science.gov (United States)

    Rand, Kasper D; Pringle, Steven D; Murphy, James P; Fadgen, Keith E; Brown, Jeff; Engen, John R

    2009-12-15

    Accumulating evidence suggests that solution-phase conformations of small globular proteins and large molecular protein assemblies can be preserved for milliseconds after electrospray ionization. Thus, the study of proteins in the gas phase on this time scale is highly desirable. Here we demonstrate that a traveling wave ion guide (TWIG) of a Synapt mass spectrometer offers a highly suitable environment for rapid and efficient gas-phase hydrogen/deuterium exchange (HDX). Gaseous ND(3) was introduced into either the source TWIG or the TWIG located just after the ion mobility cell, such that ions underwent HDX as they passed through the ND(3) on the way to the time-of-flight analyzer. The extent of deuterium labeling could be controlled by varying the quantity of ND(3) or the speed of the traveling wave. The gas-phase HDX of model peptides corresponded to labeling of primarily fast exchanging sites due to the short labeling times (ranging from 0.1 to 10 ms). In addition to peptides, gas-phase HDX of ubiquitin, cytochrome c, lysozyme, and apomyoglobin were examined. We conclude that HDX of protein ions in a TWIG is highly sensitive to protein conformation, enables the detection of conformers present on submilliseconds time scales, and can readily be combined with ion mobility spectrometry.

  18. Changes in the Factor VIII C2 domain upon membrane binding determined by hydrogen-deuterium exchange MS.

    Science.gov (United States)

    Pantazatos, Dionysios; Gessner, Christopher R; Woods, Virgil L; Gilbert, Gary E

    2014-08-01

    Factor VIII enhances the catalytic activity of Factor IXa in a membrane-bound enzyme complex and both proteins are necessary to prevent haemophilia. Tandem lectin-like C domains mediate the membrane binding of Factor VIII and membrane-interactive residues have been identified. However, the available data provide little insight into the dynamic changes that occur upon membrane binding. We used time-based hydrogen-deuterium exchange MS to evaluate the dynamics of FVIII-C2 (Factor VIII C2 domain) alone and when membrane bound. The results confirm the participation of previously identified membrane-interactive loops in the binding mechanism. In addition, they indicate that a long peptide segment, encompassing a membrane-interactive loop and strands of the β-barrel core, is remarkably dynamic prior to membrane binding. The flexibility is reduced following membrane binding. In addition, regions that interact with the A1 and C1 domains have reduced solvent exchange. Thus the isolated C2 domain has extensive flexibility that is subject to stabilization and could be related to interactions between domains as well as between Factor VIII and Factor IXa or Factor X. These results confirm that the proposed membrane-binding loops of the FVIII-C2 interact with the membrane in a manner that leads to protection from solvent exposure.

  19. Dissecting the Binding Mode of Low Affinity Phage Display Peptide Ligands to Protein Targets by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Lohse, Brian; Ming, Shonoi A

    2014-01-01

    of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, non...

  20. Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Comamala Grau, Gerard; Trelle, Morten Beck

    2016-01-01

    and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX...

  1. Oxazolone Versus Macrocycle Structures for Leu-Enkephalin b(2)-b(4): Insights from Infrared Multiple-Photon Dissociation Spectroscopy and Gas-Phase Hydrogen/Deuterium Exchange

    NARCIS (Netherlands)

    Chen, X. A.; Steill, J. D.; Oomens, J.; Pollfer, N. C.

    2010-01-01

    The collision-induced dissociation (CID) products b(2)-b(4) from Leu-enkephalin are examined with infrared multiple-photon dissociation (IR-MPD) spectroscopy and gas-phase hydrogen/deuterium exchange (HDX). Infrared spectroscopy reveals that b(2) exclusively adopts oxazolone structures, protonated

  2. Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry

    NARCIS (Netherlands)

    Bereszczak, Jessica Z; Watts, Norman R; Wingfield, Paul T; Steven, Alasdair C; Heck, Albert J R

    Hepatitis B virus core-antigen (capsid protein) and e-antigen (an immune regulator) have almost complete sequence identity, yet the dimeric proteins (termed Cp149d and Cp(-10)149d , respectively) adopt quite distinct quaternary structures. Here we use hydrogen deuterium exchange-mass spectrometry

  3. Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry

    DEFF Research Database (Denmark)

    Petersen, Michael; Madsen, Jeppe B; Jørgensen, Thomas J D

    2017-01-01

    activator inhibitor 1 (PAI-1). We report the first multi-microsecond atomistic molecular dynamics simulations of PAI-1 and compare the data with experimental hydrogen/deuterium-exchange data (HDXMS). The simulations reveal notable conformational flexibility of helices D, E and F and major fluctuations...

  4. Accessibility changes within diphtheria toxin T domain when in the functional molten globule state, as determined using hydrogen/deuterium exchange measurements

    Czech Academy of Sciences Publication Activity Database

    Man, Petr; Montagner, C.; Vitrac, H.; Kavan, Daniel; Pichard, S.; Gillet, D.; Forest, E.; Forge, V.

    2010-01-01

    Roč. 277, č. 3 (2010), s. 653-662 ISSN 1742-464X Institutional research plan: CEZ:AV0Z50200510 Keywords : diphtheria toxin * hydrogen/deuterium exchanges * mass spectrometry Subject RIV: CE - Biochemistry Impact factor: 3.129, year: 2010

  5. Radical isomerization and hydrogen--deuterium exchange in reactions of silver p-tert-butylbenzoate

    International Nuclear Information System (INIS)

    Fields, E.K.; Meyerson, S.

    1978-01-01

    Silver p-tert-butylbenzoate decomposes at 300 0 C to products that retain the tert-butyl group intact. Among these products are five isomeric di-tert-butylbiphenyls, evidently resulting by isomerization of the first-formed p-tert-butylphenyl radical. With labeled benzophenone and benzene, the silver salt gives products in which much protium--deuterium exchange has occurred. The photolyzed silver salt arylates 1,2,4-trichlorobenzene; thermal decomposition in benzonitrile yields triphenyltriazine in addition to the radical arylation product. 9 tables

  6. "TOF2H": A precision toolbox for rapid, high density/high coverage hydrogen-deuterium exchange mass spectrometry via an LC-MALDI approach, covering the data pipeline from spectral acquisition to HDX rate analysis

    Directory of Open Access Journals (Sweden)

    Koter Marek D

    2008-09-01

    Full Text Available Abstract Background Protein-amide proton hydrogen-deuterium exchange (HDX is used to investigate protein conformation, conformational changes and surface binding sites for other molecules. To our knowledge, software tools to automate data processing and analysis from sample fractionating (LC-MALDI mass-spectrometry-based HDX workflows are not publicly available. Results An integrated data pipeline (Solvent Explorer/TOF2H has been developed for the processing of LC-MALDI-derived HDX data. Based on an experiment-wide template, and taking an ab initio approach to chromatographic and spectral peak finding, initial data processing is based on accurate mass-matching to fully deisotoped peaklists accommodating, in MS/MS-confirmed peptide library searches, ambiguous mass-hits to non-target proteins. Isotope-shift re-interrogation of library search results allows quick assessment of the extent of deuteration from peaklist data alone. During raw spectrum editing, each spectral segment is validated in real time, consistent with the manageable spectral numbers resulting from LC-MALDI experiments. A semi-automated spectral-segment editor includes a semi-automated or automated assessment of the quality of all spectral segments as they are pooled across an XIC peak for summing, centroid mass determination, building of rates plots on-the-fly, and automated back exchange correction. The resulting deuterium uptake rates plots from various experiments can be averaged, subtracted, re-scaled, error-barred, and/or scatter-plotted from individual spectral segment centroids, compared to solvent exposure and hydrogen bonding predictions and receive a color suggestion for 3D visualization. This software lends itself to a "divorced" HDX approach in which MS/MS-confirmed peptide libraries are built via nano or standard ESI without source modification, and HDX is performed via LC-MALDI using a standard MALDI-TOF. The complete TOF2H package includes additional (eg LC

  7. Effects of basic site proximity on deprotonation and hydrogen/deuterium exchange reactions for model dodecapeptide ions containing lysine and glycine

    Science.gov (United States)

    Zhang, Xin; Ewing, Nigel P.; Cassady, Carolyn J.

    1998-05-01

    The effects of basic site proximity on gas-phase deprotonation and hydrogen/deuterium (H/D) exchange reactions were investigated for three model dodecapeptide ions in a Fourier transform ion cyclotron resonance mass spectrometer. Each peptide contained four high basicity lysine (K) residues and eight low basicity glycine (G) residues; however, the ordering of the residues differed. In the deprotonation studies, `fully protonated' peptide ions, [M + 4H]4+, where M = (KGG)4, (K2G4)2, and K4G8, were reacted with reference compounds of known basicities. Reaction efficiencies were in the order: [K4G8 + 4H]4+ > [(K2G4)2 + 4H]4+ ~ [(KGG)4 + 4H]4+. The facile reaction of [K4G8 + 4H]4+ is consistent with this ion having the highest Coulomb energy. For gas-phase H/D exchange reactions with d4-methanol, [K4G8 + 4H]4+ has the fastest exchange rate and undergoes the largest number of exchanges; 22 of the 26 labile hydrogens exchanged within the timescale studied. In contrast, [(KGG)4 + 4H]4+ and [(K2G4)2 + 4H]4+ reacted more slowly, but at similar rates, with a maximum of 14 observed exchanges for both ions. Molecular dynamics calculations were conducted to gain insights into conformations. In the lowest energy structures for [(KGG)4 + 4H]4+ and [(K2G4)2 + 4H]4+, the lysine n-butylamino chains stretch out to minimize Coulomb energy; there is little or no intramolecular hydrogen bonding involving the protonated amino groups. In contrast, for [K4G8 + 4H]4+, the proximity of the basicity residues makes minimization of the Coulomb energy difficult; instead, the structure becomes more compact with stabilization of the protonated amino groups by extensive intramolecular hydrogen bonding to heteroatoms in the peptide backbone. The calculated structures suggest that, in the H/D exchange reactions, the compact conformation of [K4G8 + 4H]4+ allows stabilization of the methanolpeptide intermediate by hydrogen bonding, thus lowering the barrier to proton transfer within the complex. The

  8. Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Mistarz, Ulrik H; Brown, Jeffery M; Haselmann, Kim F

    2016-01-01

    Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub...... conditions. Lysozyme ions bound by an oligosaccharide incorporated less deuterium than the unbound ion. Similarly, trypsin ions showed reduced deuterium uptake when bound by the peptide ligand vasopressin. Our results are in good agreement with crystal structures of the native protein complexes......, and illustrate that gas-phase HDX-MS can provide a sensitive and simple approach to measure the number of heteroatom-bound non-amide side-chain hydrogens involved in the binding interface of biologically relevant protein complexes....

  9. Spectroscopic, DFT, and XRD Studies of Hydrogen Bonds in N-Unsubstituted 2-Aminobenzamides

    Directory of Open Access Journals (Sweden)

    Malose Jack Mphahlele

    2017-01-01

    Full Text Available The structures of the mono- and the dihalogenated N-unsubstituted 2-aminobenzamides were characterized by means of the spectroscopic (1H-NMR, UV-Vis, FT-IR, and FT-Raman and X-ray crystallographic techniques complemented with a density functional theory (DFT method. The hindered rotation of the C(O–NH2 single bond resulted in non-equivalence of the amide protons and therefore two distinct resonances of different chemical shift values in the 1H-NMR spectra of these compounds were observed. 2-Amino-5-bromobenzamide (ABB as a model confirmed the presence of strong intramolecular hydrogen bonds between oxygen and the amine hydrogen. However, intramolecular hydrogen bonding between the carbonyl oxygen and the amine protons was not observed in the solution phase due to a rapid exchange of these two protons with the solvent and fast rotation of the Ar–NH2 single bond. XRD also revealed the ability of the amide unit of these compounds to function as a hydrogen bond donor and acceptor simultaneously to form strong intermolecular hydrogen bonding between oxygen of one molecule and the NH moiety of the amine or amide group of the other molecule and between the amine nitrogen and the amide hydrogen of different molecules. DFT calculations using the B3LYP/6-311++G(d,p basis set revealed that the conformer (A with oxygen and 2-amine on the same side predominates possibly due to the formation of a six-membered intramolecular ring, which is assisted by hydrogen bonding as observed in the single crystal XRD structure.

  10. Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, H.A.; Wang, C.; Zhao, X.; Hamuro, Y.; Conners, K.; Kearins, M.C.; Lu, F.; Sauder, J.M.; Molnar, K.S.; Coales, S.J.; Maloney, P.C.; Guggino, W.B.; Wetmore, D.R.; Weber, P.C.; Hunt, J.F. (SGX); (ExSAR); (Cystic); (JHU-MED); (Columbia)

    2012-04-30

    The {Delta}F508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and {Delta}F508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because {Delta}F508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and {Delta}F508 constructs, and the {Delta}F508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide {sup 1}H/{sup 2}H exchange rates in matched F508 and {Delta}F508 constructs reveal that {Delta}F508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the {Delta}F508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-{Delta}F508 structures but completely solvent exposed in all {Delta}F508 structures. These results reinforce the importance of the perturbation {Delta}F508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased

  11. Wet oxidative destruction of spent ion-exchange resins using hydrogen peroxide

    International Nuclear Information System (INIS)

    Srinivas, C.; Ramaswamy, M.; Theyyunni, T.K.

    1994-01-01

    Spent organic ion exchange resins are generated in large quantities during the operation of nuclear facilities. Wet oxidation as a mode of treatment of these gel-type ion exchange resins was investigated using H 2 O 2 as oxidant in the presence of CuSO 4 as catalyst. Experiments using commercial samples were conducted at 95-100 deg C under reflux conditions at atmospheric pressure. It was found that the reaction of cation resin with H 2 O 2 was instantaneous whereas with anion resin, there was a lag time. For efficient utilization of the oxidant, low rate of addition of H 2 O 2 , 0.01M concentration of CuSO 4 and neutral pH in mixed resin reactions, were found to be useful. Foaming was noted during reactions involving anion resins. This could be controlled by silicone-based agents. The residual solution left after resin oxidation is aqueous in nature and is expected to contain all the radioactivity originally present in the resin. Preliminary experiments showed that it could be efficiently trapped using available inorganic sorbents. Wet oxidation system offers a simple method of converting organic waste into environmentally acceptable inorganic products. (author). 8 refs., 6 figs., 2 tabs

  12. Use of 1,5-diaminonaphthalene to combine matrix-assisted laser desorption/ionization in-source decay fragmentation with hydrogen/deuterium exchange.

    Science.gov (United States)

    Lemaire, Pascale; Debois, Delphine; Smargiasso, Nicolas; Quinton, Loïc; Gabelica, Valérie; De Pauw, Edwin A

    2013-08-30

    In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the deuterons following in-solution hydrogen/deuterium exchange (HDX). This matrix must circumvent the commonly encountered undesired back-exchange reactions, in order to preserve the regioselective deuteration pattern. The 1,5-diaminonaphthalene (1,5-DAN) matrix is known to be suitable for MALDI-ISD fragmentation. MALDI Mass Spectrometry Imaging (MSI) was employed to compare 1,5-DAN and other commonly used MALDI matrices with respect to the extent of back-exchange and the uniformity of the H/D exchange profiles within the MALDI spots. We tested the back-exchange on the highly sensitive amyloid-beta peptide (1-40), and proved the regioselectivity on ubiquitin and β-endorphin. MALDI-MSI results show that 1,5-DAN leads to the least back-exchange over all the spot. MALDI-ISD fragmentation combined with H/D exchange using 1,5-DAN matrix was validated by localizing deuterons in native ubiquitin. Results agree with previous data obtained by Nuclear Magnetic Resonance (NMR) and Electron Transfer Dissociation (ETD). Moreover, 1,5-DAN matrix was used to study the H/D exchange profile of the methanol-induced helical structure of β-endorphin, and the relative protection can be explained by the polarity of residues involved in hydrogen bond formation. We found that controlling crystallization is the most important parameter when combining H/D exchange with MALDI. The 1,5-DAN matrix is characterized by a fast crystallization kinetics, and therefore gives robust and reliable H/D exchange profiles using MALDI-ISD. Copyright © 2013 John Wiley & Sons, Ltd.

  13. Heat Exchange and Fouling Analysis on a Set of Hydrogen Sulphide Gas Coolers

    Directory of Open Access Journals (Sweden)

    Andrés Adrian Sánchez-Escalona

    2017-07-01

    Full Text Available The sulphide acid coolers are tube and shell jacketed heat exchangers designed to cool down the produced gas from 416,15 K to 310,15 K in addition to separate the sulphur carried over by the outlet gas from the reactor tower. The investigation was carried out by applying the passive experimentation process in an online cooler set in order to determine the heat transfer rates and fouling based on heat resistance. It was corroborated that the operation of this equipment outside design parameters increases outlet gas temperature and liquid sulphur carryovers. Efficiency loss is caused by fouling elements in the fluid, which results in changes in the overall heat transfer rate. The linear tendency of the fouling heat resistance based on time for three gas flowrates.

  14. Improvements on heavy water separation technology by isotopic water-hydrogen sulfide exchange

    International Nuclear Information System (INIS)

    Peculea, M.

    1987-01-01

    A series of possible variance is presented for the heavy water separation technology by isotopic H 2 O-H 2 S exchange at dual temperatures. The critical study of these variants, which are considered as characteristic quantities for the isotopes transport (production) and the extraction level is related to a dual temperature plant fed by liquid and cold column, which is the up-to-date technology employed in all heavy water production plants as variants of following plants are studied: dual temperature plant with double feeding; dual-temperature plant with equilibrium column (booster); dual-temperature-dual-pressure plant. Attention is paid to the variant with equilibration column (booster), executed and tested at the State Committee for Nuclear Energy and to the dual-temperature-dual pressure plant which presents the highest efficiency. (author)

  15. Hydrogen-deuterium exchange in the black-eyed pea trypsin and chymotrypsin inhibitar and its complex with β-trypsin

    International Nuclear Information System (INIS)

    Mizuta, K.; Ikemoto, H.; Ventura, M.M.

    1980-01-01

    The H-D exchange of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) in D 2 O was studied by an ultraviolet spectroscopic method recently proposed (J.J. Englander, D.B. Calhoun, and S.W. Englander (1979) Analytical Biochemistry, 92, 517-524). Isotopic exchange data are presented as plots of X (The fraction of unex-changed peptide hydrogen atoms at time t) versus log(k 0 t), where K 0 is the pH dependent rate constant for peptide groups exposed to the solvent. In the range of pD2.25-6.9, at 20 0 C, BTCI shows a continuous exchange curve which indicates that the exchange mechanism is of the EX 2 type and no detectable conformational changes occur in the protein. Deviations from this exchange curve are found at pD 7.3 and 8.0. About 60% of the peptide hydrogens of BTCI are exchanged for ΔG 0 0 [pt

  16. Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.

    Directory of Open Access Journals (Sweden)

    Masaru Miyagi

    2011-02-01

    Full Text Available Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS determines the HDX rates at the imidazole C(2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR, an enzyme proposed to undergo multiple conformational changes during catalysis.Using His-HDX-MS, the pK(a values and the half-lives (t(1/2 of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX, DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH, and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+ were determined. The results showed that the two parameters (pK(a and t(1/2 are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a, t(1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a and t(1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

  17. Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.

    Science.gov (United States)

    Miyagi, Masaru; Wan, Qun; Ahmad, Md Faiz; Gokulrangan, Giridharan; Tomechko, Sara E; Bennett, Brad; Dealwis, Chris

    2011-02-16

    Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings. Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

  18. Sodium borohydride hydrogen generator using Co–P/Ni foam catalysts for 200 W proton exchange membrane fuel cell system

    International Nuclear Information System (INIS)

    Oh, Taek Hyun; Gang, Byeong Gyu; Kim, Hyuntak; Kwon, Sejin

    2015-01-01

    The response characteristics of electroless-deposited Co–P/Ni foam catalysts for sodium borohydride hydrolysis were investigated. The effect of nickel foam geometry on the properties of the catalysts was evaluated. As the PPI (pores per inch) of the nickel foam increased, the hydrogen generation rate per gram of the deposited catalyst increased due to an increase in surface area. The response characteristics of various catalysts were compared under real operating conditions. When a thin nickel foam with high PPI was used, the response characteristics of the catalyst improved due to an increase in the amount of the deposited catalyst and surface area. Finally, a 200 W PEMFC (proton exchange membrane fuel cell) system using electroless-deposited Co–P/Ni foam (110 PPI) catalyst was investigated. The response time to reach a hydrogen generation rate sufficient for a 200 W PEMFC was 71 s, and the energy density of a 200 W fuel cell system for producing 600 Wh was 252.1 Wh/kg. A fuel cell system using Co–P/Ni foam catalysts can be widely used as a power source for mobile applications due to fast response characteristics and high energy density. - Highlights: • Response characteristics of Co–P/Ni foam catalysts are investigated. • Catalytic activity is improved with increase in PPI (pores per inch) of Ni foam. • Co–P/Ni foam (110 PPI) catalyst has improved response characteristics. • The energy density of a 200 W PEMFC system for producing 600 Wh is 252.1 Wh/kg. • Co–P/Ni foam (110 PPI) catalyst is suitable for fuel cell system.

  19. Zoniporide: a potent and selective inhibitor of the human sodium-hydrogen exchanger isoform 1 (NHE-1).

    Science.gov (United States)

    Tracey, W Ross; Allen, Mary C; Frazier, Donald E; Fossa, Anthony A; Johnson, Celeste G; Marala, Ravi B; Knight, Delvin R; Guzman-Perez, Angel

    2003-01-01

    The sodium-hydrogen exchanger isoform-1 (NHE-1) plays an important role in the myocardial response to ischemia-reperfusion; inhibition of this exchanger protects against ischemic injury, including reduction in infarct size. Herein we describe a novel, potent, and highly selective NHE-1 inhibitor, zoniporide (CP-597,396; [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine). Zoniporide inhibits human NHE-1 with an IC(50) of 14 nM, has >150-fold selectivity vs. other NHE isoforms, and potently inhibits ex vivo NHE-1-dependent swelling of human platelets. This compound is well tolerated in preclinical animal models, exhibits moderate plasma protein binding, has a t(1/2) of 1.5 h in monkeys, and has one major active metabolite. In both in vitro and in vivo rabbit models of myocardial ischemia-reperfusion injury, zoniporide markedly reduced infarct size without adversely affecting hemodynamics or cardiac function. In the isolated heart (Langendorff), zoniporide elicited a concentration-dependent reduction in infarct size (EC(50) = 0.25 nM). At 50 nM it reduced infarct size by 83%. This compound was 2.5-20-fold more potent than either eniporide or cariporide (EC(50)s of 0.69 and 5.11 nM, respectively), and reduced infarct size to a greater extent than eniporide. In open chest, anesthetized rabbits, zoniporide also elicited a dose-dependent reduction in infarct size (ED(50) = 0.45 mg/kg/h) and inhibited NHE-1-mediated platelet swelling (93% inhibition at 4 mg/kg/h). Furthermore, zoniporide attenuated postischemic cardiac contractile dysfunction in conscious primates, and reduced both the incidence and duration of ischemia-reperfusion-induced ventricular fibrillation in rats. Zoniporide represents a novel class of potent and selective human NHE-1 inhibitors with potential utility for providing cardioprotection in a clinical setting.

  20. Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange.

    Science.gov (United States)

    Chien, Chih-Ta Henry; Ho, Meng-Ru; Lin, Chung-Hung; Hsu, Shang-Te Danny

    2017-08-16

    Galectins are β-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8 NTD and hGal8 CTD , respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8 NTD . We further demonstrated using the NMR-based hydrogen-deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8 NTD , indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8 CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8 CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy.

  1. Online Hydrogen-Deuterium Exchange Traveling Wave Ion Mobility Mass Spectrometry (HDX-IM-MS): a Systematic Evaluation

    Science.gov (United States)

    Cryar, Adam; Groves, Kate; Quaglia, Milena

    2017-06-01

    Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an important tool for measuring and monitoring protein structure. A bottom-up approach to HDX-MS provides peptide level deuterium uptake values and a more refined localization of deuterium incorporation compared with global HDX-MS measurements. The degree of localization provided by HDX-MS is proportional to the number of peptides that can be identified and monitored across an exchange experiment. Ion mobility spectrometry (IMS) has been shown to improve MS-based peptide analysis of biological samples through increased separation capacity. The integration of IMS within HDX-MS workflows has been commercialized but presently its adoption has not been widespread. The potential benefits of IMS, therefore, have not yet been fully explored. We herein describe a comprehensive evaluation of traveling wave ion mobility integrated within an online-HDX-MS system and present the first reported example of UDMSE acquisition for HDX analysis. Instrument settings required for optimal peptide identifications are described and the effects of detector saturation due to peak compression are discussed. A model system is utilized to confirm the comparability of HDX-IM-MS and HDX-MS uptake values prior to an evaluation of the benefits of IMS at increasing sample complexity. Interestingly, MS and IM-MS acquisitions were found to identify distinct populations of peptides that were unique to the respective methods, a property that can be utilized to increase the spatial resolution of HDX-MS experiments by >60%. [Figure not available: see fulltext.

  2. Mechanism of Lipid Binding of Human Apolipoprotein E3 by Hydrogen/Deuterium Exchange/Mass Spectrometry and Fluorescence Polarization.

    Science.gov (United States)

    Fabilane, Charina S; Nguyen, Patricia N; Hernandez, Roy V; Nirudodhi, Sasidhar; Duong, Mai; Maier, Claudia S; Narayanaswami, Vasanthy

    2016-01-01

    Human apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in maintaining plasma cholesterol/triglyceride homeostasis. The C-terminal (CT) domain of apoE3 (residues 201-299) is composed of amphipathic α-helices C1: W210-S223, C2: V236-E266, and C3: D271-W276, which play a dominant role in mediating high-affinity lipid binding. The objective is to understand the accessibility of the CT domain at the sub-domain level and the mechanistic details regarding lipid-binding interaction. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX/MS) of recombinant wild type (WT) apoE(201-299), chemical-induced unfolding monitored as changes in fluorescence polarization (FP) of labeled apoE(201-299) bearing a probe at specified sites, and lipid binding studies were carried out. HDX/MS revealed that residues towards the C-terminal end of the domain display significantly lower %D uptake compared to those towards the center, suggesting extensive protein-protein interaction in this segment. Functional assays showed that locking apoE(201-299) in an inter-molecular disulfide-bonded state at position 209, 223, 255, or 277 significantly decreases its ability to interact with lipids, especially when tethered towards the ends; this could be restored by reduction. Unfolding studies indicate that the C-terminal end offers less resistance to unfolding compared to the central portion of the domain. Taken together, our data suggest that two dimers of CT domain are juxtaposed around helix C3 leading to apoE3 tetramerization, and that dissociation to monomeric units is a required step in lipid binding, with helix C3 likely seeking stability via lipid interaction prior to helices C1 or C2.

  3. [Secondary Structure of Aβ(1-16) Complexes with Zinc: A Study in the Gas Phase Using Deuterium/Hydrogen Exchange and Ultra-High-Resolution Mass Spectrometry].

    Science.gov (United States)

    Kostyukevich, Yu I; Kononikhin, A S; Indeykina, M I; Popov, I A; Bocharov, K V; Spassky, A I; Kozin, S A; Makarov, A A; Nikolaev, E N

    2017-01-01

    Complexes of peptide fragment 1-16 of beta-amyloid with transition metals play an important role in the development of a broad class of neurodegenerative diseases, which determines the interest in investigating the structures of these complexes. In this work, we have applied the method of the deuterium/hydrogen exchange in combination with ultra-high-resolution mass spectrometry to study conformational changes in (1-16) beta-amyloid peptide induced by binding of zinc(II) atoms. The efficiency of the deuterium/hydrogen exchange depended on the number of zinc atoms bound to the peptide and on the temperature of the ionization source region. Deuterium/hydrogen exchange reactions have been performed directly in the ionization source. The number of exchanges decreased considerably with an increasing numbers of zinc atoms. The relationship has been described with a damped exponential curve, which indicated that the binding of zinc atoms altered the conformation of the peptide ion by making it less open, which limits the access to inner areas of the molecule.

  4. Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

    Science.gov (United States)

    White, Mark A; Li, Sheng; Tsalkova, Tamara; Mei, Fang C; Liu, Tong; Woods, Virgil L; Cheng, Xiaodong

    2012-01-01

    Exchange proteins directly activated by cAMP (EPACs) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

  5. Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

    Directory of Open Access Journals (Sweden)

    Mark A White

    Full Text Available Exchange proteins directly activated by cAMP (EPACs are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

  6. A novel sodium-hydrogen exchanger isoform-1 inhibitor, zoniporide, reduces ischemic myocardial injury in vitro and in vivo.

    Science.gov (United States)

    Knight, D R; Smith, A H; Flynn, D M; MacAndrew, J T; Ellery, S S; Kong, J X; Marala, R B; Wester, R T; Guzman-Perez, A; Hill, R J; Magee, W P; Tracey, W R

    2001-04-01

    The cardioprotective efficacy of zoniporide (CP-597,396), a novel, potent, and selective inhibitor of the sodium-hydrogen exchanger isoform 1 (NHE-1), was evaluated both in vitro and in vivo using rabbit models of myocardial ischemia-reperfusion injury. In these models, myocardial injury was elicited with 30 min of regional ischemia and 120 min of reperfusion. Zoniporide elicited a concentration-dependent reduction in infarct size (EC(50) of 0.25 nM) in the isolated heart (Langendorff) and reduced infarct size by 83% (50 nM). This compound was 2.5- to 20-fold more potent than either eniporide or cariporide (EC(50) of 0.69 and 5.11 nM, respectively), and reduced infarct size to a greater extent than eniporide (58% reduction in infarct size). In open-chest, anesthetized rabbits, zoniporide also elicited a dose-dependent reduction in infarct size (ED(50) of 0.45 mg/kg/h) and inhibited NHE-1-mediated platelet swelling (maximum inhibition 93%). Furthermore, zoniporide did not cause any in vivo hemodynamic (mean arterial pressure, heart rate, rate pressure product) changes. Zoniporide represents a novel class of potent NHE-1 inhibitors with potential utility for providing clinical cardioprotection.

  7. Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Houde, Damian; Nazari, Zeinab E.; Bou-Assaf, George M.; Weiskopf, Andrew S.; Rand, Kasper D.

    2016-04-01

    When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein-protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, "traditional" continuous dilution labeling HDX-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied this approach to a highly concentrated antibody solution without dilution or significant sample manipulation, prior to analysis. Such a method could pave the way for a deeper understanding of the unusual behavior of proteins at high concentrations, which is highly relevant for development of biopharmaceuticals in industry.

  8. Applications of hydrogen deuterium exchange (HDX for the characterization of conformational dynamics in light-activated photoreceptors

    Directory of Open Access Journals (Sweden)

    Robert eLindner

    2015-06-01

    Full Text Available Rational design of optogenetic tools is inherently linked to the understanding of photoreceptor function. Structural analysis of elements involved in signal integration in individual sensor domains provides an initial idea of their mode of operation, but understanding how local structural rearrangements eventually affect signal transmission to output domains requires inclusion of the effector regions in the characterization. However, the dynamic nature of these assemblies renders their structural analysis challenging and therefore a combination of high- and low-resolution techniques is required to appreciate functional aspects of photoreceptors.This review focuses on the potential of Hydrogen-Deuterium exchange coupled to mass spectrometry (HDX-MS for complementing the structural characterization of photoreceptors. In this respect, the ability of HDX-MS to provide information on the conformational dynamics and the possibility to address multiple functionally relevant states in solution render this methodology ideally suitable. We highlight recent examples demonstrating the potential of HDX-MS and discuss how these results can help to improve existing optogenetic systems or guide the design of novel optogenetic tools.

  9. Activity-regulating structural changes and autoantibody epitopes in transglutaminase 2 assessed by hydrogen/deuterium exchange.

    Science.gov (United States)

    Iversen, Rasmus; Mysling, Simon; Hnida, Kathrin; Jørgensen, Thomas J D; Sollid, Ludvig M

    2014-12-02

    The multifunctional enzyme transglutaminase 2 (TG2) is the target of autoantibodies in the gluten-sensitive enteropathy celiac disease. In addition, the enzyme is responsible for deamidation of gluten peptides, which are subsequently targeted by T cells. To understand the regulation of TG2 activity and the enzyme's role as an autoantigen in celiac disease, we have addressed structural properties of TG2 in solution by using hydrogen/deuterium exchange monitored by mass spectrometry. We demonstrate that Ca(2+) binding, which is necessary for TG2 activity, induces structural changes in the catalytic core domain of the enzyme. Cysteine oxidation was found to abolish these changes, suggesting a mechanism whereby disulfide bond formation inactivates the enzyme. Further, by using TG2-specific human monoclonal antibodies generated from intestinal plasma cells of celiac disease patients, we observed that binding of TG2 by autoantibodies can induce structural changes that could be relevant for the pathogenesis. Detailed mapping of two of the main epitopes targeted by celiac disease autoantibodies revealed that they are located adjacent to each other in the N-terminal part of the TG2 molecule.

  10. Dynamics and ligand-induced conformational changes in human prolyl oligopeptidase analyzed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Tsirigotaki, Alexandra; Elzen, Roos Van; Veken, Pieter Van Der; Lambeir, Anne-Marie; Economou, Anastassios

    2017-05-26

    Prolyl oligopeptidase (PREP) is conserved in many organisms across life. It is involved in numerous processes including brain function and neuropathology, that require more than its strict proteolytic role. It consists of a seven-bladed β-propeller juxtaposed to a catalytic α/β-hydrolase domain. The conformational dynamics of PREP involved in domain motions and the gating mechanism that allows substrate accessibility remain elusive. Here we used Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) to derive the first near-residue resolution analysis of global PREP dynamics in the presence or absence of inhibitor bound in the active site. Clear roles are revealed for parts that would be critical for the activation mechanism. In the free state, the inter-domain interface is loose, providing access to the catalytic site. Inhibitor binding "locks" the two domains together exploiting prominent interactions between the loop of the first β-propeller blade and its proximal helix from the α/β-hydrolase domain. Loop A, thought to drive gating, is partially stabilized but remains flexible and dynamic. These findings provide a conformational guide for further dissection of the gating mechanism of PREP, that would impact drug development. Moreover, they offer a structural framework against which to study proteolysis-independent interactions with disordered proteins like α-synuclein involved in neurodegenerative disease.

  11. The exchange reaction between hydrogen and deuterium. II - Proposal for an heterogeneous initiation mechanism of gaseous phase reactions

    International Nuclear Information System (INIS)

    Marteau, Chantal; Gaillard-Cusin, Francoise; James, Henri

    1978-01-01

    Investigation of experimental data related to evolution period exhibited by H 2 -D 2 exchange process requires to take into account the variation against time of every atomic species -adsorbed or not- implied in the reaction mechanism. The formation of first chain carriers involves: - chemisorption of either gaseous reactant on the surface active centres (Σ), e.g.: Σ + 1/2 H 2 reversible ΣH; - consecutive generation of atomic species through hetero-homogeneous transfer between chemisorbed species (ΣH) and gaseous molecules: ΣH+H 2 →Σ+H 2 +H 0 , ΣH+D 2 →Σ+HD+D 0 . Therefore, it can be shown that the heterogeneous initiation process of a gas phase reaction identifies to a chain linear mechanism. Such an heterogeneous sequence conditions the further proceeding of the homogeneous chain reaction; both evolutions being kinematically connected. Rate constant of hydrogen adsorption on silica glass: ksub(a1) approximately 10 14 exp(-47/RT)Isup(0,5).molesup(-0,5).S -1 has been evaluated [fr

  12. HCN elimination from vinyl cyanide: product energy partitioning, the role of hydrogen-deuterium exchange reactions and a new pathway.

    Science.gov (United States)

    Vázquez, Saulo A; Martínez-Núñez, Emilio

    2015-03-14

    The different HCN elimination pathways from vinyl cyanide (VCN) are studied in this paper using RRKM, Kinetic Monte Carlo (KMC), and quasi-classical trajectory (QCT) calculations. A new HCN elimination pathway proves to be very competitive with the traditional 3-center and 4-center mechanisms, particularly at low excitation energies. However, low excitation energies have never been experimentally explored, and the high and low excitation regions are dynamically different. The KMC simulations carried out using singly deuterated VCN (CH2=CD-CN) at 148 kcal mol(-1) show the importance of hydrogen-deuterium exchange reactions: both DCN and HCN will be produced in any of the 1,1 and 1,2 elimination pathways. The QCT simulation results obtained for the 3-center pathway are in agreement with the available experimental results, with the 4-center results showing much more excitation of the products. In general, our results seem to be consistent with a photodissociation mechanism at 193 nm, where the molecule dissociates (at least the HCN elimination pathways) in the ground electronic state. However, our simulations assume that internal conversion is a fully statistical process, i.e., the HCN elimination channels proceed on the ground electronic state according to RRKM theory, which might not be the case. In future studies it would be of interest to include the photo-prepared electronically excited state(s) in the dynamics simulations.

  13. Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization.

    Science.gov (United States)

    Dautant, Alain; Meyer, Philippe; Georgescauld, Florian

    2017-06-13

    Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the wild type (WT) and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding and/or folding by hydrogen/deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are nativelike dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.

  14. Influence of domain interactions on conformational mobility of the progesterone receptor detected by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Goswami, Devrishi; Callaway, Celetta; Pascal, Bruce D; Kumar, Raj; Edwards, Dean P; Griffin, Patrick R

    2014-07-08

    Structural and functional details of the N-terminal activation function 1 (AF1) of most nuclear receptors are poorly understood due to the highly dynamic intrinsically disordered nature of this domain. A hydrogen/deuterium exchange (HDX) mass-spectrometry-based investigation of TATA box-binding protein (TBP) interaction with various domains of progesterone receptor (PR) demonstrate that agonist-bound PR interaction with TBP via AF1 impacts the mobility of the C-terminal AF2. Results from HDX and other biophysical studies involving agonist- and antagonist-bound full-length PR and isolated PR domains reveal the molecular mechanism underlying synergistic transcriptional activation mediated by AF1 and AF2, dominance of PR-B isoform over PR-A, and the necessity of AF2 for full AF1-mediated transcriptional activity. These results provide a comprehensive picture elaborating the underlying mechanism of PR-TBP interactions as a model for studying nuclear receptor (NR)-transcription factor functional interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Drug binding and resistance mechanism of KIT tyrosine kinase revealed by hydrogen/deuterium exchange FTICR mass spectrometry.

    Science.gov (United States)

    Zhang, Hui-Min; Yu, Xiu; Greig, Michael J; Gajiwala, Ketan S; Wu, Joe C; Diehl, Wade; Lunney, Elizabeth A; Emmett, Mark R; Marshall, Alan G

    2010-04-01

    Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution-phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT-KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild-type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants.

  16. Coarse-Grained Conformational Sampling of Protein Structure Improves the Fit to Experimental Hydrogen-Exchange Data.

    Science.gov (United States)

    Devaurs, Didier; Antunes, Dinler A; Papanastasiou, Malvina; Moll, Mark; Ricklin, Daniel; Lambris, John D; Kavraki, Lydia E

    2017-01-01

    Monitoring hydrogen/deuterium exchange (HDX) undergone by a protein in solution produces experimental data that translates into valuable information about the protein's structure. Data produced by HDX experiments is often interpreted using a crystal structure of the protein, when available. However, it has been shown that the correspondence between experimental HDX data and crystal structures is often not satisfactory. This creates difficulties when trying to perform a structural analysis of the HDX data. In this paper, we evaluate several strategies to obtain a conformation providing a good fit to the experimental HDX data, which is a premise of an accurate structural analysis. We show that performing molecular dynamics simulations can be inadequate to obtain such conformations, and we propose a novel methodology involving a coarse-grained conformational sampling approach instead. By extensively exploring the intrinsic flexibility of a protein with this approach, we produce a conformational ensemble from which we extract a single conformation providing a good fit to the experimental HDX data. We successfully demonstrate the applicability of our method to four small and medium-sized proteins.

  17. Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

    DEFF Research Database (Denmark)

    Trelle, Morten Beck; Hirschberg, Daniel; Jansson, Anna

    2012-01-01

    and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected...... against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1...

  18. A membrane cell for on-line hydrogen/deuterium exchange to study protein folding and protein-protein interactions by mass spectrometry.

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-09-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.

  19. A Membrane Cell for On-line Hydrogen/Deuterium Exchange to Study Protein Folding and Protein-Protein Interactions by Mass Spectrometry*

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-01-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation. PMID:21610101

  20. Study on the influence of isotope exchange of hydrogen with deuterium on the vibrational spectrum of lysozyme by inelastic neutron scattering

    International Nuclear Information System (INIS)

    Svanidze, A. V.; Lushnikov, S. G.; Sashin, I. L.; Gvasaliya, S. N.

    2007-01-01

    The influence of isotope exchange of hydrogen with deuterium on the lysozyme dynamics was studied by incoherent inelastic neutron scattering. The generalized vibrational densities of states G(ω) were constructed from experimental results for protonated and deuterated protein samples at 200, 280, and 311 K. The major isotope effect was observed in G(ω) in the frequency region higher than 100 cm -1 . At all temperatures, both the Debye mode and the region of G(ω), whose spectral dimension corresponds to the fracton mode, are observed in the low-frequency region of the densities of states of both protonated and deuterated lysozyme. The influence of the hydrogen isotope exchange on the low-frequency region of G(ω) is insignificant