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1.
J Am Chem Soc ; 144(23): 10230-10240, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35647706

RESUMO

Lanthipeptide synthetases construct macrocyclic peptide natural products by catalyzing an iterative cascade of post-translational modifications. Class II lanthipeptide synthetases (LanM enzymes) catalyze multiple rounds of peptide dehydration and thioether macrocycle formation in a manner that guides precursor peptide maturation to the biologically active final product with high fidelity. The mechanistic details underlying the contradictory phenomena of substrate flexibility coupled with high biosynthetic fidelity have proven challenging to illuminate. In this work, we employ mass spectrometry to investigate how the structure of a maturing precursor lanthipeptide (HalA2) influences the local and global structure of its cognate lanthipeptide synthetase (HalM2). Using enzymatically synthesized HalA2 peptides that contain sets of native thioether macrocycles, we employ ion mobility mass spectrometry (IM-MS) to show that HalA2 macrocyclization alters the conformational landscape of the HalM2 enzyme in a systematic manner. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies show that local HalM2 structural dynamics also change in response to HalA2 post-translational modification. Notably, deuterium uptake in a critical HalM2 α-helical region depends on the number of thioether macrocycles present in the HalA2 core peptide. Binding of the isolated leader and core peptide portions of the modular HalA2 precursor led to a synergistic structuring of this α-helical region, providing evidence for distinct leader and core peptide binding sites that independently alter the dynamics of this functionally critical α-helix. The data support a mechanistic model where the sequential post-translational modification of HalA2 alters the conformational dynamics of HalM2 in regions of the enzyme that are known to be functionally critical.


Assuntos
Bacteriocinas , Ligases , Bacteriocinas/química , Deutério , Ligases/metabolismo , Peptídeos , Sulfetos
2.
Commun Biol ; 5(1): 588, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35705679

RESUMO

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a technique to explore differential protein structure by examining the rate of deuterium incorporation for specific peptides. This rate will be altered upon structural perturbation and detecting significant changes to this rate requires a statistical test. To determine rates of incorporation, HDX-MS measurements are frequently made over a time course. However, current statistical testing procedures ignore the correlations in the temporal dimension of the data. Using tools from functional data analysis, we develop a testing procedure that explicitly incorporates a model of hydrogen deuterium exchange. To further improve statistical power, we develop an empirical Bayes version of our method, allowing us to borrow information across peptides and stabilise variance estimates for low sample sizes. Our approach has increased power, reduces false positives and improves interpretation over linear model-based approaches. Due to the improved flexibility of our method, we can apply it to a multi-antibody epitope-mapping experiment where current approaches are inapplicable due insufficient flexibility. Hence, our approach allows HDX-MS to be applied in more experimental scenarios and reduces the burden on experimentalists to produce excessive replicates. Our approach is implemented in the R-package "hdxstats": https://github.com/ococrook/hdxstats .


Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Teorema de Bayes , Deutério/química , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Peptídeos
3.
Front Immunol ; 13: 859964, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35720345

RESUMO

Although computational structure prediction has had great successes in recent years, it regularly fails to predict the interactions of large protein complexes with residue-level accuracy, or even the correct orientation of the protein partners. The performance of computational docking can be notably enhanced by incorporating experimental data from structural biology techniques. A rapid method to probe protein-protein interactions is hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS has been increasingly used for epitope-mapping of antibodies (Abs) to their respective antigens (Ags) in the past few years. In this paper, we review the current state of HDX-MS in studying protein interactions, specifically Ab-Ag interactions, and how it has been used to inform computational structure prediction calculations. Particularly, we address the limitations of HDX-MS in epitope mapping and techniques and protocols applied to overcome these barriers. Furthermore, we explore computational methods that leverage HDX-MS to aid structure prediction, including the computational simulation of HDX-MS data and the combination of HDX-MS and protein docking. We point out challenges in interpreting and incorporating HDX-MS data into Ab-Ag complex docking and highlight the opportunities they provide to build towards a more optimized hybrid method, allowing for more reliable, high throughput epitope identification.


Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Complexo Antígeno-Anticorpo , Deutério , Medição da Troca de Deutério/métodos , Epitopos , Espectrometria de Massas/métodos , Proteínas/metabolismo
4.
J Chem Phys ; 156(19): 194506, 2022 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-35597634

RESUMO

Glyceline, a green solvent considered for various electrochemical applications, represents a multi-component glass former. Viewed from this perspective, the choline cation and the hydrogen bond donor glycerol, the two major constituents forming this deep eutectic solvent, were studied using nuclear magnetic resonance in a selective manner by means of suitably deuteron-labeled isotopologues. Carried out from far above to far below the glass transition temperature, measurements and analyses of the spin-lattice and spin-spin relaxation times reveal that the reorientational dynamics of the components, i.e., of glycerol as well as of chain deuterated choline chloride are slightly different. Possible implications of this finding regarding the hydrogen-bonding pattern in glyceline are discussed. Furthermore, the deuterated methyl groups in choline chloride are exploited as sensitive probes of glyceline's supercooled and glassy states. Apart from spin relaxometry, a detailed line shape analysis of the CD3 spectra yields valuable insights into the broad intermolecular and intramolecular energy barrier distributions present in this binary mixture.


Assuntos
Colina , Glicerol , Colina/química , Deutério , Glicerol/química , Espectroscopia de Ressonância Magnética , Solventes/química
5.
J Am Chem Soc ; 144(22): 9570-9575, 2022 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-35613457

RESUMO

Deuterated organic compounds have become increasingly important in many areas; however, it remains challenging to install deuterium site-selectively to unactivated aliphatic positions with control of the degree of deuteration. Here, we report a Cu-catalyzed degree-controlled deacylative deuteration of diverse alkyl groups with the methylketone (acetyl) moiety as a traceless activating group. The use of N-methylpicolino-hydrazonamide (MPHA) promotes efficient aromatization-driven C-C cleavage. Mono-, di-, and trideuteration at specific sites can be selectively achieved. The reaction is redox-neutral with broad functional group tolerance. The utility of this method has been demonstrated in forming a complete set of deuterated ethyl groups, merging with the Diels-Alder reaction, a net devinylative deuteration, and the synthesis of the d2-analogue of Austedo.


Assuntos
Compostos Orgânicos , Catálise , Deutério , Oxirredução
6.
Neuroimage ; 257: 119284, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35533826

RESUMO

Deuterium metabolic imaging (DMI) and hyperpolarized 13C-pyruvate MRI (13C-HPMRI) are two emerging methods for non-invasive and non-ionizing imaging of tissue metabolism. Imaging cerebral metabolism has potential applications in cancer, neurodegeneration, multiple sclerosis, traumatic brain injury, stroke, and inborn errors of metabolism. Here we directly compare these two non-invasive methods at 3 T for the first time in humans and show how they simultaneously probe both oxidative and non-oxidative metabolism. DMI was undertaken 1-2 h after oral administration of [6,6'-2H2]glucose, and 13C-MRI was performed immediately following intravenous injection of hyperpolarized [1-13C]pyruvate in ten and nine normal volunteers within each arm respectively. DMI was used to generate maps of deuterium-labelled water, glucose, lactate, and glutamate/glutamine (Glx) and the spectral separation demonstrated that DMI is feasible at 3 T. 13C-HPMRI generated maps of hyperpolarized carbon-13 labelled pyruvate, lactate, and bicarbonate. The ratio of 13C-lactate/13C-bicarbonate (mean 3.7 ± 1.2) acquired with 13C-HPMRI was higher than the equivalent 2H-lactate/2H-Glx ratio (mean 0.18 ± 0.09) acquired using DMI. These differences can be explained by the route of administering each probe, the timing of imaging after ingestion or injection, as well as the biological differences in cerebral uptake and cellular physiology between the two molecules. The results demonstrate these two metabolic imaging methods provide different yet complementary readouts of oxidative and reductive metabolism within a clinically feasible timescale. Furthermore, as DMI was undertaken at a clinical field strength within a ten-minute scan time, it demonstrates its potential as a routine clinical tool in the future.


Assuntos
Bicarbonatos , Imageamento por Ressonância Magnética , Bicarbonatos/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Isótopos de Carbono/metabolismo , Deutério/metabolismo , Glucose/metabolismo , Humanos , Ácido Láctico/metabolismo , Imageamento por Ressonância Magnética/métodos , Ácido Pirúvico
7.
J Am Chem Soc ; 144(19): 8504-8514, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35508077

RESUMO

Alkyne-tagged Raman probes have shown high promise for noninvasive and sensitive visualization of small biomolecules to understand their functional roles in live cells. However, the potential for alkynes to sense cellular environments that goes beyond imaging remains to be further explored. Here, we report a general strategy for Raman imaging-based local environment sensing by hydrogen-deuterium exchange (HDX) of terminal alkynes (termed alkyne-HDX). We first demonstrate, in multiple Raman probes, that deuterations of the alkynyl hydrogens lead to remarkable shifts of alkyne Raman peaks for about 130 cm-1, providing resolvable signals suited for imaging-based analysis with high specificity. Both our analytical derivation and experimental characterizations subsequently establish that HDX kinetics are linearly proportional to both alkyne pKas and environmental pDs. After validating the quantitative nature of this strategy, we apply alkyne-HDX to sensing local chemical and cellular environments. We establish that alkyne-HDX exhibits high sensitivity to various DNA structures and demonstrates the capacity to detect DNA structural changes in situ from UV-induced damage. We further show that this strategy is also applicable to resolve subtle pD variations in live cells. Altogether, our work lays the foundation for utilizing alkyne-HDX strategy to quantitatively sense the local environments for a broad spectrum of applications in complex biological systems.


Assuntos
Alcinos , Análise Espectral Raman , Alcinos/química , DNA , Deutério , Medição da Troca de Deutério , Hidrogênio , Indicadores e Reagentes , Análise Espectral Raman/métodos
8.
J Am Soc Mass Spectrom ; 33(5): 813-822, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35385652

RESUMO

Experimental measurement of time-dependent spontaneous exchange of amide protons with deuterium of the solvent provides information on the structure and dynamical structural variation in proteins. Two experimental techniques are used to probe the exchange: NMR, which relies on different magnetic properties of hydrogen and deuterium, and MS, which exploits the change in mass due to deuteration. NMR provides residue-specific information, that is, the rate of exchange or, analogously, the protection factor (i.e., the unitless ratio between the rate of exchange for a completely unstructured state and the observed rate). MS provides information that is specific to peptides obtained by proteolytic digestion. The spatial resolution of HDX-MS measurements depends on the proteolytic pattern of the protein, the fragmentation method used, and the overlap between peptides. Different computational approaches have been proposed to extract residue-specific information from peptide-level HDX-MS measurements. Here, we demonstrate the advantages of a method recently proposed that exploits self-consistency and classifies the possible sets of protection factors into a finite number of alternative solutions compatible with experimental data. The degeneracy of the solutions can be reduced (or completely removed) by exploiting the additional information encoded in the shape of the isotopic envelopes. We show how sparse and noisy MS data can provide high-resolution protection factors that correlate with NMR measurements probing the same protein under the same conditions.


Assuntos
Medição da Troca de Deutério , Hidrogênio , Deutério/química , Medição da Troca de Deutério/métodos , Hidrogênio/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Peptídeos/química , Proteínas/química
9.
Spectrochim Acta A Mol Biomol Spectrosc ; 276: 121217, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35427921

RESUMO

Metabolic dynamics of bacterial cells is needed for understanding the correlation between changes in environmental conditions and cell metabolic activity. In this study, Raman spectroscopy combined with deuterium labelling was used to analyze the metabolic activity of a single Escherichia coli O157:H7 cell. The incorporation of deuterium from heavy water into cellular biomolecules resulted in the formation of carbon-deuterium (CD) peaks in the Raman spectra, indicating the cell metabolic activity. The broad vibrational peaks corresponding to CD and CH peaks encompassed different specific shifts of macromolecules such as protein, lipids, and nucleic acid. The utilization of tryptophan and oleic acid by the cell as the sole carbon source led to changes in cell lipid composition, as indicated by new peaks in the second derivative spectra. Thus, the proposed method could semi-quantitatively determine total metabolic activity, macromolecule specific identification, and lipid and protein metabolism in a single cell.


Assuntos
Carbono , Escherichia coli , Carbono/metabolismo , Deutério , Escherichia coli/metabolismo , Lipídeos , Substâncias Macromoleculares/metabolismo
10.
Molecules ; 27(8)2022 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-35458603

RESUMO

This review is giving a short introduction to the techniques used to investigate isotope effects on NMR chemical shifts. The review is discussing how isotope effects on chemical shifts can be used to elucidate the importance of either intra- or intermolecular hydrogen bonding in ionic liquids, of ammonium ions in a confined space, how isotope effects can help define dimers, trimers, etc., how isotope effects can lead to structural parameters such as distances and give information about ion pairing. Tautomerism is by advantage investigated by isotope effects on chemical shifts both in symmetric and asymmetric systems. The relationship between hydrogen bond energies and two-bond deuterium isotope effects on chemical shifts is described. Finally, theoretical calculations to obtain isotope effects on chemical shifts are looked into.


Assuntos
Ligação de Hidrogênio , Deutério/química , Espectroscopia de Ressonância Magnética/métodos
11.
Molecules ; 27(8)2022 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-35458625

RESUMO

Blood levels of the vitamin D3 (D3) metabolites 25-hydroxyvitamin D3 (25(OH)D3), 24R,25-dihydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are recognized indicators for the diagnosis of bone metabolism-related diseases, D3 deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D3 metabolites, such as 20-hydroxyvitamin D3 and lactone D3, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D3 metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D3 (2), 25(OH)D3-23,26-lactone (6), and 1,25(OH)2D3-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)2D3-23,26-lactone (7) in human serum.


Assuntos
Espectrometria de Massas em Tandem , Vitamina D , Cromatografia Líquida/métodos , Deutério , Humanos , Lactonas , Espectrometria de Massas em Tandem/métodos , Vitamina D/metabolismo
12.
Anal Bioanal Chem ; 414(13): 3875-3884, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35389096

RESUMO

C-Reactive protein (CRP) is an important marker for in vitro diagnosis (IVD) of inflammation. However, CRP immunoturbidimetric kits from different manufacturers exhibit inconsistency in evaluation, making clinical diagnosis challenging. The use of immunological methods in diagnosis means that the differences in epitopes across kits may directly lead to inconsistent results. Therefore, to provide consistent results, it is essential to perform epitope mapping of different kits. The composition of antibodies in a single kit is typically complex, with a combination of polyclonal antibodies or monoclonal antibodies. Here, we show an epitope screening strategy for complex antibodies in a kit based on hydrogen-deuterium exchange mass spectrometry (HDX-MS). We applied this workflow to successfully map the epitopes for three kits from three different manufacturers and compared their quantitative results. We obtained different quantitative results using kits from different manufacturers upon epitope mapping, confirming the correlation between the quantitative results and the epitopes. Thus, we have established a workflow based on HDX-MS to screen epitopes in IVD kits. This work helps determine the quantitative accuracy of a kit based on structural information, can guide the design and production of IVD reagents, and further improves the accuracy of IVD.


Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteína C-Reativa , Deutério , Medição da Troca de Deutério/métodos , Mapeamento de Epitopos/métodos , Epitopos
13.
Angew Chem Int Ed Engl ; 61(25): e202202779, 2022 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-35411582

RESUMO

We describe a concise and reliable protocol for the precisely controlled tetradeuteration of straight-chain fatty acids (FAs) at the α- and ß-positions that is generally applicable to a variety of FAs, including trans-FAs, polyunsaturated FAs (PUFAs), and their oxidized derivatives. The precisely controlled introduction of four deuterium atoms into the FAs enables their persistent and quantitative tracking by LC-MS/MS analysis based on their molecular structures. In addition, the phosphatidylcholine (PC) species prepared from the tetradeuterated FAs thus obtained give a diagnostic peak, namely, a phosphocholine fragment that contains deuterium, in the LC-MS/MS analysis. With these features, the metabolism of a representative oxidized linoleic acid, that is, hydroxyoctadecadienoic acid (HODE), was investigated, leading to the identification of acyltransferases that transfer the acyl moiety derived from HODE to lysophosphatidylcholine.


Assuntos
Ácidos Graxos , Ácido Linoleico , Cromatografia Líquida , Deutério , Ácidos Linoleicos/química , Espectrometria de Massas em Tandem
14.
Anal Biochem ; 652: 114675, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35390328

RESUMO

Inclusion bodies (IBs) are large, insoluble aggregates that often form during the overexpression of proteins in bacteria. These aggregates are of broad fundamental and practical significance, for recombinant protein preparation and due to their relevance to aggregation-related medical conditions and their recent emergence as promising functional nanomaterials. Despite their significance, high resolution knowledge of IB structure remains very limited. Such knowledge will advance understanding and control of IB formation and properties in myriad practical applications. Here, we report a detailed quenched hydrogen-deuterium amide exchange (qHDX) method with NMR readout to define the structure of IBs at the level of individual residues throughout the protein. Applying proper control of experimental conditions, such as sample pH, water content, temperature, and intrinsic rate of amide exchange, yields in depth results for these cellular protein aggregates. qHDX results illustrated for Cu, Zn superoxide dismutase 1 (SOD1) and Adnectins show their IBs include native-like structure and some but not all mutations alter IB structure.


Assuntos
Hidrogênio , Síndrome do Intestino Irritável , Amidas/química , Deutério/química , Humanos , Hidrogênio/química , Agregados Proteicos , Proteínas
15.
J Pharm Biomed Anal ; 215: 114754, 2022 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-35427962

RESUMO

Monoclonal antibody (mAb) has grown to be the major asset in protein therapeutics market since its initial introduction in 1980s. To identify a suitable mAb as a drug candidate for development requires deep understanding of disease biology and attribute sciences, including epitope-paratope binding recognition. Mass spectrometry (MS) has become a critical technology platform in epitope mapping. MS-based approaches utilize chemical labeling to assess the changes of solvent accessibilities during binding interactions, and the labeling can be either reversible or irreversible. Reversible labeling is represented by hydrogen/deuterium exchange (HDX), which probes the changes via exchange between backbone amide hydrogen and deuterium in the solvent. Irreversible labeling targets amino acid residue side chains and involves chemical based labeling such as glycine ethyl ester labeling, radical based labeling such as fast photochemical oxidation of proteins (FPOP), and chemical cross-linking. All these methods have been developed extensively for characterization of binding interface within an immunocomplex. This review covers the fundamentals of these different MS-based methods and highlights recent case studies to illustrate unique capabilities of MS-based approaches in epitope mapping of protein therapeutics.


Assuntos
Anticorpos Monoclonais , Medição da Troca de Deutério , Anticorpos Monoclonais/química , Deutério , Medição da Troca de Deutério/métodos , Mapeamento de Epitopos/métodos , Espectrometria de Massas/métodos , Solventes
16.
Langmuir ; 38(18): 5515-5524, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35477243

RESUMO

The chain melting of lipid bilayers has often been investigated in detail using calorimetric methods, such as differential scanning calorimetry (DSC), and the resultant main transition temperature is regarded as one of the most important parameters in model membrane experiments. However, it is not always clear whether the hydrocarbon chains of lipids are gradually melting along the depth of the lipid bilayer or whether they all melt concurrently in a very narrow temperature range, as implied by DSC. In this study, we focused on stearoyl-d-sphingomyelin (SSM) as an example of raft-forming lipids. We synthesized deuterium-labeled SSMs at the 4', 10', and 16' positions, and their depth-dependent melting was measured using solid-state deuterium NMR by changing the temperature by 1.0 °C, and comparing with that observed from a saturated lipid, palmitoylstearoylphosphatidylcholine (PSPC). The results showed that SSM exhibited a characteristic depth-dependent melting, which was not observed for PSPC. The strong intermolecular hydrogen bonds between the sphingomyelin amide moiety probably caused the chain melting to start from the chain terminus through the middle part and end in the upper part. This depth-dependent melting implies that the small gel-like domains of SSM remain at temperatures slightly above the main transition temperature. These sphingomyelin features may be responsible for the biological properties of SM-based lipid rafts.


Assuntos
Bicamadas Lipídicas , Esfingomielinas , Varredura Diferencial de Calorimetria , Deutério , Bicamadas Lipídicas/química , Microdomínios da Membrana , Fosfatidilcolinas/química , Esfingomielinas/química , Temperatura
17.
J Am Chem Soc ; 144(16): 7327-7336, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35416652

RESUMO

Deuterated amino acids have been recognized for their utility in drug development, for facilitating nuclear magnetic resonance (NMR) analysis, and as probes for enzyme mechanism. Small molecule-based methods for the site-selective synthesis of deuterated amino acids typically involve de novo synthesis of the compound from deuterated precursors. In comparison, enzymatic methods for introducing deuterium offer improved efficiency, operating directly on free amino acids to achieve hydrogen-deuterium (H/D) exchange. However, site selectivity remains a significant challenge for enzyme-mediated deuteration, limiting access to desirable deuteration motifs. Here, we use enzyme-catalyzed deuteration, combined with steady-state kinetic analysis and ultraviolet (UV)-vis spectroscopy to probe the mechanism of a two-protein system responsible for the biosynthesis of l-allo-Ile. We show that an aminotransferase (DsaD) can pair with a small partner protein (DsaE) to catalyze Cα and Cß H/D exchange of amino acids, while reactions without DsaE lead exclusively to Cα-deuteration. With conditions for improved catalysis, we evaluate the substrate scope for Cα/Cß-deuteration and demonstrate the utility of this system for preparative-scale, selective labeling of amino acids.


Assuntos
Aminas , Aminoácidos , Catálise , Deutério/química , Hidrogênio/química , Cinética , Proteínas
18.
Sci Rep ; 12(1): 6351, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35428795

RESUMO

The doubly labelled water (DLW) method is widely used to determine energy expenditure. In this work, we demonstrate the addition of the third stable isotope, 17O, to turn it into triply labelled water (TLW), using the three isotopes measurement of optical spectrometry. We performed TLW (2H, 18O and17O) measurements for the analysis of the CO2 production (rCO2) of mice on different diets for the first time. Triply highly enriched water was injected into mice, and the isotope enrichments of the distilled blood samples of one initial and two finals were measured by an off-axis integrated cavity output spectroscopy instrument. We evaluated the impact of different calculation protocols and the values of evaporative water loss fraction. We found that the dilution space and turnover rates of 17O and 18O were equal for the same mice group, and that values of rCO2 calculated based on 18O-2H, or on 17O-2H agreed very well. This increases the reliability and redundancy of the measurements and it lowers the uncertainty in the calculated rCO2 to 3% when taking the average of two DLW methods. However, the TLW method overestimated the rCO2 compared to the indirect calorimetry measurements that we also performed, much more for the mice on a high-fat diet than for low-fat. We hypothesize an extra loss or exchange mechanism with a high fractionation for 2H to explain this difference.


Assuntos
Dióxido de Carbono , Água , Animais , Deutério/análise , Metabolismo Energético , Camundongos , Isótopos de Oxigênio/análise , Reprodutibilidade dos Testes
19.
Nucleic Acids Res ; 50(7): 4042-4053, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35380691

RESUMO

Mtr4 is a eukaryotic RNA helicase required for RNA decay by the nuclear exosome. Previous studies have shown how RNA en route to the exosome threads through the highly conserved helicase core of Mtr4. Mtr4 also contains an arch domain, although details of potential interactions between the arch and RNA have been elusive. To understand the interaction of Saccharomyces cerevisiae Mtr4 with various RNAs, we have characterized RNA binding in solution using hydrogen-deuterium exchange mass spectrometry, and affinity and unwinding assays. We have identified RNA interactions within the helicase core that are consistent with existing structures and do not vary between tRNA, single-stranded RNA and double-stranded RNA constructs. We have also identified novel RNA interactions with a region of the arch known as the fist or KOW. These interactions are important for RNA unwinding and vary in strength depending on RNA structure and length. They account for Mtr4 discrimination between different RNAs. These interactions further drive Mtr4 to adopt a closed conformation characterized by reduced dynamics of the arch arm and intra-domain contacts between the fist and helicase core.


Assuntos
RNA Helicases DEAD-box/química , Proteínas de Saccharomyces cerevisiae/química , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Deutério/metabolismo , Medição da Troca de Deutério , Espectrometria de Massas , RNA/genética , RNA/metabolismo , RNA Helicases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Chem Commun (Camb) ; 58(26): 4215-4218, 2022 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35274648

RESUMO

Herein, a tunable iodization/deuterolysis protocol for phosphonium ylides by employing D2O as the deuterium source was designed. Notably, this process could be manipulated by tuning the base, thus leading to two valuable deuterated building blocks - benzyl iodides and aromatic aldehydes with broad substrate scope, good functional group compatibility and excellent deuteration degree. Concise syntheses of a series of deuterated drug analogues have been achieved based on the developed deuteration reaction platform.


Assuntos
Aldeídos , Iodetos , Deutério
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