Levels Of Deuterium Incorporation Research Articles

Changes in protein structure monitored by use of gas-phase hydrogen/deuterium exchange.

The study of protein conformation by solution‐phase hydrogen/deuterium exchange (HDX) coupled to MS is well documented. This involves monitoring the exchange of backbone amide protons with deuterium and provides details concerning the protein's tertiary structure. However, undesired back‐exchange during post‐HDX analyses can be difficult to control. Here, gas‐phase HDX‐MS, during which labile hydrogens on amino acid side chains are exchanged in sub‐millisecond time scales, has been employed to probe changes within protein structures. Addition of the solvent 2,2,2‐trifluoroethanol to a protein in solution can affect the structure of the protein, resulting in an increase in secondary and/or tertiary structure which is detected using circular dichroism. Using a Synapt G2‐S ESI‐mass spectrometer modified to allow deuterated ammonia into the transfer ion guide (situated between the ion mobility cell and the TOF analyser), gas‐phase HDX‐MS is shown to reflect minor structural changes experienced by the proteins β‐lactoglobulin and ubiquitin, as observed by the reduction in the level of deuterium incorporation. Additionally, the use of gas‐phase HDX‐MS to distinguish between co‐populated proteins conformers within a solution is demonstrated with the disordered protein calmodulin; the gas‐phase HDX‐MS results correspond directly with complementary data obtained by use of ion mobility spectrometry‐MS.

Production of bacterial cellulose with controlled deuterium-hydrogen substitution for neutron scattering studies.

Isotopic enrichment of biomacromolecules is a widely used technique that enables the investigation of the structural and dynamic properties to provide information not accessible with natural abundance isotopic composition. This study reports an approach for deuterium incorporation into bacterial cellulose. A media formulation for growth of Acetobacter xylinus subsp. sucrofermentans and Gluconacetobacter hansenii was formulated that supports cellulose production in deuterium (D) oxide. The level of D incorporation can be varied by altering the ratio of deuterated and protiated glycerol used during cell growth in the D2O-based growth medium. Spectroscopic analysis and mass spectrometry show that the level of deuterium incorporation is high (>90%) for the perdeuterated form of bacterial cellulose. The small-angle neutron scattering profiles of the cellulose with different amounts of D incorporation are all similar indicating that there are no structural changes in the cellulose due to substitution of deuterium for hydrogen. In addition, by varying the amount of deuterated glycerol in the media it was possible to vary the scattering length density of the deuterated cellulose. The ability to control deuterium content of cellulose extends the range of experiments using techniques such as neutron scattering to reveal information about the structure and dynamics of cellulose, and its interactions with other biomacromolecules as well as synthetic polymers used for development of composite materials.

Controlled incorporation of deuterium into bacterial cellulose

Isotopic enrichment has been widely used for investigating the structural and dynamic properties of biomacromolecules to provide information that cannot be carried out with molecules composed of natural abundance isotopes. A media formulation for controlled incorporation of deuterium in bacterial cellulose synthesized by Gluconacetobacter xylinus subsp. sucrofermentans is reported. The purified cellulose was characterized using Fourier Transform Infra-Red spectrophotometry and mass spectrometry which revealed that the level of deuterium incorporation in the perdeuterated cellulose was greater than 90 %. Small-angle neutron scattering analysis demonstrated that the overall structure of the cellulose was unaffected by the substitution of deuterium for hydrogen. In addition, by varying the amount of D-glycerol in the media it was possible to vary the scattering length density of the deuterated cellulose. A large disk model was used to fit the curves of bacterial cellulose grown using 0 and 100 % D-Glycerol yielding a lower bound to the disk radii, R min = 1,132 ± 6 and 1,154 ± 3 A and disk thickness, T = 128 ± 1 and 83 ± 1 A for the protiated and deuterated forms of the bacterial cellulose, respectively. This agrees well with the scanning electron microscopy analysis which revealed stacked sheets in the cellulose pellicles. Controlled incorporation of deuterium into cellulose will enable new types of experiments using techniques such as neutron scattering to reveal information about the structure and dynamics of cellulose and its interactions with proteins and other (bio) polymers.

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein beta-2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of beta-2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the beta-sheet-rich insoluble aggregates of beta-2-microglobulin.

The effect of adding Crabtree's catalyst to rhodium black in direct hydrogen isotope exchange reactions

AbstractA new catalytic system based on rhodium black using Crabtree's catalyst as an additive for direct hydrogen isotope exchange in aromatic compounds has been investigated. The level of deuterium incorporation can be improved from for example 16 to 93%. The new catalyst mixture tolerates a variety of solvents. Copyright © 2009 John Wiley & Sons, Ltd.

Influence of [2H]-labelled acetic acid as solvent in the synthesis of [2H]-labelled perhexiline

Preparation of deuterium-labelled perhexiline from an unsaturated analogue was performed via reduction with deuterium gas and PtO2 in acetic acid. Low incorporation was observed when using acetic acid as solvent (most abundant mass peak was M), but when changing the solvent to deuterium-labelled acetic acid, e.g. acetic acid-OD or acetic acid-d4, a higher incorporation was observed (most abundant mass peak was M). Using hydrogen gas instead of deuterium gas with deuterium-labelled acetic acid, high levels of deuterium incorporation were observed (most abundant mass peak was M). An attempt to reduce a precursor with a fully deuterated pyridine to obtain perhexiline with a higher content of deuterium failed. Copyright © 2009 John Wiley & Sons, Ltd.

Efficient and practical syntheses of a stable labelled form of the non‐narcotic analgesic, bicifadine and its metabolites

AbstractAn efficient and practical synthesis of deuterium‐labelled bicifadine, together with 3 deuterium‐labelled bicifadine metabolites, was developed. The levels of deuterium incorporation are high, and the products were used as internal standards in bioanalytical method development and clinical bioanalysis. Copyright © 2007 John Wiley & Sons, Ltd.

An efficient laboratory synthesis of α-deuteriated profens

An efficient and practical laboratory synthesis of a series of 2-deuterio-2-arylpropionic acids (α-deuterioprofens) is described. The levels of deuterium incorporation are high and the products are synthetically useful. Copyright © 2006 John Wiley & Sons, Ltd.

Use of amide exchange mass spectrometry to study conformational changes within the endopolygalacturonase II-homogalacturonan-polygalacturonase inhibiting protein system.

Amide exchange mass spectrometry (MS) was used to study the enzyme endopolygalacturonase II (EPG-II) from Aspergillus niger as it binds to an oligosaccharide substrate. A localized decrease in the level of deuterium incorporation in EPG-II of the EPG-II-oligosaccharide complex relative to that of the free EPG-II identified the location of substrate contact, which is in agreement with published site specific mutation studies. In addition, when bound with substrate, regions of EPG-II remote from the substrate binding site became exposed to the solvent, as revealed by an increase in the amount of incorporated deuterium, indicating a conformational change in the enzyme. Fluorescence experiments were performed to provide additional evidence for an altered conformation of EPG-II as a result of substrate binding. This novel application of amide exchange-MS to the study of protein-carbohydrate binding has, for the first time, described in detail the conformational changes associated with EPG-II when it binds a substrate. Amide exchange-MS was also used to study the interactions of EPG-II and the polygalacturonase inhibitor protein (PGIP). Mass spectral data of the EPG-II-oligosaccharide complex in the presence of Phaseolus vulgaris PGIP indicate that the inhibitor contacts EPG-II at a site remote from the substrate binding cleft, and is restricting the conformational changes of EPG-II. Fluorescence experiments also revealed that upon binding of PGIP, the conformational changes mentioned above for the EPG-II-substrate complex are minimized. These results, together with previously reported data, point to a location on EPG-II for interaction with PGIP as well as a possible mechanism for noncompetitive inhibition of EPG-II.

Borohydride exchange resin as an alternative method for deuterium labelling: selective reduction of aryl bromides

AbstractTransition metal‐activated borohydride exchange resin and borodeuteride exchange resin have been prepared and used for the selective reductive deuteration of aryl bromides. This methodology, which utilizes deuterated methanol as the primary source of label, provides an alternative method for the introduction of deuterium. Investigation of which bond in the solvent donates the deuterium and model reductions on ortho‐bromo‐N,N‐dimethylbenzamide suggest that an organocopper(III) intermediate is most likely involved. The immobilized hydride on resin also provides an easy workup and gives products of high purity. This methodology is far more selective than typical catalytic deuteration and, in most cases, provides products with high levels of deuterium incorporation. Copyright © 2001 John Wiley & Sons, Ltd.

Efficient synthesis of deuterium- and tritium-labeledd-erythro-sphingosine

Deuterium- and tritium-labeled d-erythro-sphingosine (1b and 1c) were prepared by an efficient approach based on a known stereoselective total synthesis. Tritium was introduced at C−1 by [3H]NaBH4 reduction in the final synthetic step to give 1c of high radiochemical purity. 1,1,3-Trideuterio-d-erythro-sphingosine (1b) was prepared similarly and showed >99·5% enantiomeric purity and a high level of deuterium incorporation. Copyright © 1999 John Wiley & Sons, Ltd.