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1.
Proc Natl Acad Sci U S A ; 117(22): 11916-11922, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414932

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both [Formula: see text] and [Formula: see text] as cosubstrates. In this study, the [Formula: see text] reaction with reduced Hypocrea jecorina LPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than the [Formula: see text] reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze-quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O-O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuII sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.


Assuntos
Aminoácidos/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/química , Polissacarídeos/metabolismo , Sítios de Ligação , Biocombustíveis , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hypocrea/metabolismo , Cinética , Espectroscopia de Ressonância Magnética/métodos , Oxigenases de Função Mista/metabolismo , Oxirredução , Peróxidos/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo
2.
PLoS One ; 15(5): e0232555, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392255

RESUMO

We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Animais , Espectroscopia de Ressonância de Spin Eletrônica/instrumentação , Congelamento , Cavalos , Cinética , Micro-Ondas , Mioglobina/química
3.
Biochim Biophys Acta Proteins Proteom ; 1868(6): 140413, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32179182

RESUMO

Only recently it was discovered that haemoglobin (Hb) belongs to the standard gene repertoire of insects, although their tracheal system is used for respiration. A classical oxygen-carrying function of Hb is only obvious for hexapods living in hypoxic environments. In other insect species, including the common fruit fly Drosophila melanogaster, the physiological role of Hb is yet unclear. Here, we study recombinant haemoglobin from the European honeybee Apis mellifera (Ame) and the malaria mosquito Anopheles gambiae (Aga). Spectroscopic evidence shows that both proteins can be classified as hexacoordinate Hbs with a strong affinity for the distal histidine. AgaHb1 is proposed to play a role in oxygen transport or sensing based on its multimeric state, slow autoxidation, and small but significant amount of five-coordinated haem in the deoxy ferrous form. AmeHb appears to behave more like vertebrate neuroglobin with a complex function given its diversified distribution in the genome.


Assuntos
Anopheles/metabolismo , Abelhas/metabolismo , Hemoglobinas/análise , Sistema Respiratório/metabolismo , Análise Espectral/métodos , Animais , Anopheles/genética , Abelhas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Evolução Molecular , Compostos Férricos/química , Compostos Ferrosos/química , Genoma , Heme/metabolismo , Hemoglobinas/genética , Insetos/genética , Insetos/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Oxigênio
4.
Arch Biochem Biophys ; 684: 108323, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126206

RESUMO

Electron Paramagnetic Resonance is a spectroscopic technique which, in combination with site-directed spin-labeling, provides structural and dynamic information about proteins in conditions similar to those of their physiological environment. The information is sequence-resolved, as it is based on probing the local dynamics of a paramagnetic label incorporated as a side chain of a selected amino acid. EPR does not impose a limit on the size of the protein or protein complex, as long as it is amenable to site-directed mutagenesis, and is able to obtain reliable distance distributions between two or more labels (identical or different).. The mean value, width and shape of distance distributions, as well as their dependence upon the state of the protein or interactions with physiological partners, provide insight into order-disorder transitions and the roles of protein flexibility. The main potentialities and limitations of the technique are revised and illustrated with examples of proteins for which order-disorder play an important role.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas Intrinsicamente Desordenadas/química , Marcadores de Spin , Óxidos N-Cíclicos/química , Cisteína/química , Maleabilidade , Conformação Proteica
5.
Chem Pharm Bull (Tokyo) ; 68(2): 150-154, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009082

RESUMO

Singlet oxygen (1O2) is highly oxidative and exerts strong cytotoxic effects. We tried to establish the best combination of a singlet oxygen generation system and a detection method with ESR, for measurement of the quenching activities of various substances. The photosensitizing reaction of rose bengal or thermal decomposition of 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide, EP) was used for the generation of 1O2, and a sterically hindered secondary amine, 2,2,6,6-tetramethyl-4-piperidone (TEMPD) or 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH), was used as the 1O2 detection probe. These secondary amines were oxidized by 1O2 to form stable nitroxide radicals, which were detectable by ESR. TEMPD was found to be readily oxidized by air, causing large background signals in comparison with TEMP-OH. The ESR signal obtained by the irradiation of rose bengal with visible light in the presence of TEMP-OH consisted of two kinds of nitroxide radical overlapping. In contrast, only a single nitroxide signal was observed when TEMP-OH was reacted with 1O2 generated from EP. Therefore, the best combination should be EP as the 1O2 generator and TEMP-OH as the detection probe. When using this combination, we found that the concentrations of some organic solvents such as dimethyl sulfoxide and acetonitrile should be kept constant for reliable quantification, because the concentrations of organic solvents affect the ESR signal intensity.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Oxigênio Singlete/análise , Oxirredução , Fármacos Fotossensibilizantes/química , Piperidonas/química , Propionatos/química , Rosa Bengala/química
6.
Food Chem ; 315: 126237, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32014666

RESUMO

Lipid derived free radical in soymilks were studied by combining 5,5-dimethyl-pyrroline-l-oxide (DMPO) spin trap, chloroform-methanol extraction and electron spin resonance (ESR) spectroscopy. Five lipid derived free radical adducts: DMPO-X, DMPO-L, DMPO-R, DMPO-LOO, and DMPO-RO were presented in soymilks. The total amounts of spins increased as the soaking temperature increased from 4 °C to 50 °C and the soaking pH increased from 3 to 9 and in paralleled with the diffusion of soybean exudates to soaking water. Prolonged soaking of soybean at 50 °C resulted in a higher signal intensity of DMPO-R than that of DMPO-LOO. Soybean lipoxygenases (LOXs) were responsible for the formation of lipid derived free radicals in soymilks. Soybean exudates affected the total amounts of lipid radicals in linoleic acid (LA) - LOX model system. The relative signal intensities of DMPO-R and DMPO-LOO were depended on the contents of soybean exudates in the system.


Assuntos
Radicais Livres/química , Lipídeos/química , Leite de Soja/química , Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Concentração de Íons de Hidrogênio , Ácido Linoleico/química , Soja/química , Marcadores de Spin , Temperatura
7.
J Oleo Sci ; 69(1): 1-6, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902891

RESUMO

X-band (9.4 GHz) electron paramagnetic resonance (EPR) and electron paramagnetic resonance imaging (EPRI) were used for elucidating structural aspects of the stratum corneum (SC). We found that psoriasis vulgaris (PV)-SC has a less-ordered structure than that of the control SC, indicating the abnormal architecture of PV-SC. Different spectra were observed for the PV-SC. The three-line spectral pattern suggests that the 5-doxylstearic acid (5-DSA) is mobile or less rigid in the SC. The simulated order parameter (S0) value obtained for 5-DSA in the SC was approximately 0.20. The statistical analysis suggests that the value of the PV-SC is significantly smaller than that of the control (p < 0.01). Thus, we suggest that this EPR assay is of great use for evaluating SC function. In addition, the EPRI of various SC samples provides a useful image concerning SC status. A strong red image was observed for the PV skin. No red lesion region was observed in the control. The EPR images differentiated various sizes and number distribution concerning the disordered states in the SC.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas de Filamentos Intermediários/química
8.
Proc Natl Acad Sci U S A ; 117(5): 2441-2448, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964841

RESUMO

Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions. We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein.


Assuntos
Membrana Celular/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas de Membrana/química , Anticorpos de Domínio Único/química , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Materiais Biocompatíveis , Membrana Celular/metabolismo , Gadolínio/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único/metabolismo , Marcadores de Spin
9.
Biochim Biophys Acta Biomembr ; 1862(5): 183207, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987867

RESUMO

Amphiphilic maleic acid-containing copolymers account for a recent methodical breakthrough in the study of membrane proteins. Their application enables a detergent-free extraction of membrane proteins from lipid bilayers, yielding stable water-soluble, discoidal lipid bilayer particles with incorporated proteins, which are wrapped with copolymers. Although many studies confirm the potential of this approach for membrane protein research, the interactions between the maleic acid-containing copolymers and extracted lipids, as well as possible effects of the copolymers on lipid-embedded proteins deserve further scrutinization. Here, we combine electron paramagnetic resonance spectroscopy and coarse-grain molecular dynamics simulations to compare the distribution and dynamics of lipids in lipid particles of phospholipid bilayers encased either by an aliphatic diisobutylene/maleic acid copolymer (DIBMALPs) or by an aromatic styrene/maleic acid copolymer (SMALPs). Nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels experience restrictions of their reorientational motion depending on the type of encasing copolymer. The dynamics of the lipids was less constrained in DIBMALPs than in SMALPs with the affinity of spin labeled lipids to the polymeric rim being more pronounced in SMALPs.


Assuntos
Bicamadas Lipídicas/química , Maleatos/química , Nanopartículas/química , Alcenos/química , Dimiristoilfosfatidilcolina/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Fosfolipídeos , Polímeros/química , Poliestirenos/química , Marcadores de Spin/síntese química
10.
Biochim Biophys Acta Gen Subj ; 1864(2): 129332, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-30928492

RESUMO

We describe the recent progress of structural analysis methods exploiting paramagnetic lanthanide ions. In NMR spectroscopy, the paramagnetic effects induced by the trivalent lanthanide ions provide long-range (~40 Å) distance and angular information that can be exploited in protein structure determination, ligand screening, structure-based resonance assignment, and in-cell observation. The paramagnetic lanthanide ions can also be utilized in EPR spectroscopy, providing nanometer-scale distance measurement. These applications of the paramagnetic lanthanide probe are becoming more widespread by the use of a variety of lanthanide binding tags. Here, we introduce the basics of paramagnetic effects, several examples of lanthanide tags, and recent applications of paramagnetic lanthanide ions in NMR and EPR spectroscopy. Collectively, we show how the paramagnetic lanthanide probe accelerates research in protein science and drug design, and consequently life science.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Elementos da Série dos Lantanídeos/química , Cisteína/química , Ácido Edético/química , Escherichia coli , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas/química
11.
Proc Natl Acad Sci U S A ; 117(1): 141-145, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31848244

RESUMO

Knowledge of the manganese oxidation states of the oxygen-evolving Mn4CaO5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S2 state of this cluster. This signal implies an overall oxidation state of either Mn(III)3Mn(IV) or Mn(III)Mn(IV)3 for the S2 state. These 2 competing assignments are commonly known as "low oxidation (LO)" and "high oxidation (HO)" models of the Mn4CaO5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn4CaO5 cluster starting from Mn(II) and Mn-free PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn4CaO5 cluster's oxidation states.


Assuntos
Manganês/metabolismo , Oxigênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Cianobactérias , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Lasers , Luz , Compostos de Manganês , Modelos Químicos , Oxirredução , Óxidos , Complexo de Proteína do Fotossistema II/química , Água/química
12.
Respir Physiol Neurobiol ; 271: 103309, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31561012

RESUMO

Pulmonary surfactant main function is to reduce surface tension at alveolar interface. Two lipids phases coexist in surfactant membranes: a liquid-ordered (Lo) and a liquid-disordered (Ld) phases. This coexistence of phases would be crucial for the surfactant activity. Until now, the proportion of phases was determined qualitatively. We design an electronic spin resonance technique to quantify the lipid fraction in Ld phase. An exogenous pulmonary surfactant (EPS) with or without extra Cho was labeled with 5-doxil stearic acid to estimate the membrane fluidity and with TEMPO to determine the PL in Ld phase. A unique equation was established for the calculation of PL in Ld phase with an error of less than 3%. TEMPO partition coefficient was (0.78 ±â€¯0.03). Cholesterol added to EPS did not modify this coefficient. The equation is valid for different batches of surfactant regardless of the cholesterol content. The proposed method is simple, precise and allows evaluating changes in lateral structure that could affect surfactant biophysical properties.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Surfactantes Pulmonares/análise , Surfactantes Pulmonares/química , Animais , Bovinos , Colesterol/análise , Colesterol/química , Tensão Superficial
13.
Biomed Microdevices ; 22(1): 3, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31797058

RESUMO

Electron paramagnetic resonance (EPR) spectroscopy using oxygen-sensing implants can provide reliable and repeated measurements of the partial pressure of oxygen (pO2) over a period of months or longer; however, it does not provide accurate information about the distribution of tissue oxygenation. While EPR imaging has the capability to provide spatially resolved oxygen data, it is time-consuming and not optimized for discrete number of implants. Previous reports suggest multi-site algorithms, which would require either the implants to be aligned in a certain way so as to deconvolve them using a linear magnetic field gradient or sparse imaging of the implants from a small number of 3D projections. In this paper, we present a simpler and much faster method to estimate the pO2 histogram from a composite, single-scan EPR spectrum measured without applying field gradients to separate the EPR signals from multiple randomly placed oxygen-sensing implants. The method is optimized for a discrete number of implants, validated using simulations, experimental phantoms and in animal models. The results established the composite spectral fitting algorithm as a reliable and robust tool for multi-site oximetry.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Oxigênio/metabolismo , Pressão , Próteses e Implantes , Limite de Detecção
14.
Nat Commun ; 10(1): 4619, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601809

RESUMO

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Cisteína/genética , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Canais Iônicos/genética , Bicamadas Lipídicas/química , Lipídeos/química , Mutação , Conformação Proteica , Domínios Proteicos
15.
Proc Natl Acad Sci U S A ; 116(39): 19458-19463, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31488720

RESUMO

Photosystem II (PSII), the light-driven water/plastoquinone photooxidoreductase, is of central importance in the planetary energy cycle. The product of the reaction, plastohydroquinone (PQH2), is released into the membrane from the QB site, where it is formed. A plastoquinone (PQ) from the membrane pool then binds into the QB site. Despite their functional importance, the thermodynamic properties of the PQ in the QB site, QB, in its different redox forms have received relatively little attention. Here we report the midpoint potentials (Em ) of QB in PSII from Thermosynechococcus elongatus using electron paramagnetic resonance (EPR) spectroscopy: Em QB/QB •- ≈ 90 mV, and Em QB •-/QBH2 ≈ 40 mV. These data allow the following conclusions: 1) The semiquinone, QB •-, is stabilized thermodynamically; 2) the resulting Em QB/QBH2 (∼65 mV) is lower than the Em PQ/PQH2 (∼117 mV), and the difference (ΔE ≈ 50 meV) represents the driving force for QBH2 release into the pool; 3) PQ is ∼50× more tightly bound than PQH2; and 4) the difference between the Em QB/QB •- measured here and the Em QA/QA •- from the literature is ∼234 meV, in principle corresponding to the driving force for electron transfer from QA •- to QB The pH dependence of the thermoluminescence associated with QB •- provided a functional estimate for this energy gap and gave a similar value (≥180 meV). These estimates are larger than the generally accepted value (∼70 meV), and this is discussed. The energetics of QB in PSII are comparable to those in the homologous purple bacterial reaction center.


Assuntos
Benzoquinonas/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Quinonas/metabolismo , Cianobactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Transporte de Elétrons , Cinética , Luz , Oxirredução , Fotossíntese/fisiologia , Plastoquinona/análogos & derivados , Plastoquinona/metabolismo , Termodinâmica , Água/química
16.
Phys Chem Chem Phys ; 21(34): 18699-18705, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31423504

RESUMO

Fully hydrated bilayers of monounsaturated palmitoyloleoylphosphatidylcholine (POPC) and diunsaturated dioleoylphosphatidylcholine (DOPC) lipids have low main phase transition temperatures (271 K for POPC and 253 K for DOPC). Two-pulse echo detected spectra, combined with continuous wave electron paramagnetic resonance spectroscopy, are employed to study the low-temperature lamellar phases of the POPC and DOPC unsaturated bilayers that are usually studied in the fluid state. Phosphatidylcholine spin-labeled at C-5 and C-16 carbon atom positions along the acyl chain were used and the temperature varied over the range 77-270 K. Segmental chain librational oscillations of small amplitude and with correlation time in the subnanosecond to nanosecond range are found in both membranes. The mean-square angular amplitude, α2, of librations increases with temperature, is larger close to the bilayer midplane than close to the first acyl chain segments, and is larger in diunsaturated than in monounsaturated bilayers. In the inner hydrocarbon region of both lipid matrices, α2 increases first slowly and linearly with temperature and then more rapidly, and a dynamical transition is detected in the range 190-210 K. Compared to dipalmitoylphosphatidylcholine bilayers of fully saturated symmetric chain lipids, the presence of double bonds in the acyl chain enhances the intensity of librational motion which is characterized by larger angular variations at the terminal methyl ends. These findings highlight biophysical properties of unsaturated bilayers in the frozen state, including a detailed characterization of segmental chain dynamics and the evidence of a dynamical transition that appears to be a generic feature in hydrated macromolecular systems. These results can also be relevant in regulating membrane physical properties and function at higher physiological temperatures.


Assuntos
Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Cinética , Conformação Molecular , Movimento (Física) , Transição de Fase , Fosfatidilcolinas/química , Marcadores de Spin , Temperatura , Termodinâmica
17.
Nat Protoc ; 14(9): 2707-2747, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31451784

RESUMO

Nitrogen-vacancy (NV) quantum defects in diamond are sensitive detectors of magnetic fields. Owing to their atomic size and optical readout capability, they have been used for magnetic resonance spectroscopy of nanoscale samples on diamond surfaces. Here, we present a protocol for fabricating NV diamond chips and for constructing and operating a simple, low-cost 'quantum diamond spectrometer' for performing NMR and electron spin resonance (ESR) spectroscopy in nanoscale volumes. The instrument is based on a commercially available diamond chip, into which an NV ensemble is ion-implanted at a depth of ~10 nm below the diamond surface. The spectrometer operates at low magnetic fields (~300 G) and requires standard optical and microwave (MW) components for NV spin preparation, manipulation, and readout. We demonstrate the utility of this device for nanoscale proton and fluorine NMR spectroscopy, as well as for the detection of transition metals via relaxometry. We estimate that the full protocol requires 2-3 months to implement, depending on the availability of equipment, diamond substrates, and user experience.


Assuntos
Diamante/química , Espectroscopia de Ressonância de Spin Eletrônica/instrumentação , Espectroscopia de Ressonância Magnética/instrumentação , Nanotecnologia/instrumentação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Espectroscopia de Ressonância Magnética/métodos , Nanopartículas/química , Processamento de Sinais Assistido por Computador
18.
Cell Biochem Biophys ; 77(3): 187-196, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31440878

RESUMO

This paper presents a survey of electron paramagnetic resonance (EPR) image registration. Image registration is the process of overlaying images (two or more) of the same scene taken at different times, from different viewpoints and/or different techniques. EPR-imaging (EPRI) techniques belong to the functional-imaging modalities and therefore suffer from a lack of anatomical reference which is mandatory in preclinical imaging. For this reason, it is necessary to merging EPR images with other modalities which allow for obtaining anatomy images. Methodological analysis and review of the literature were done, providing a summary for developing a good foundation for research study in this field which is crucial in understanding the existing levels of knowledge. Out of these considerations, the aim of this paper is to enhance the scientific community's understanding of the current status of research in EPR preclinical image registration and also communicate to them the contribution of this research in the field of image processing.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Animais , Processamento de Imagem Assistida por Computador , Articulação do Joelho/diagnóstico por imagem , Imagem por Ressonância Magnética , Camundongos
19.
Anal Bioanal Chem ; 411(25): 6677-6686, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31372701

RESUMO

The antioxidant capacity of 22 kinds of fruits was measured by the developed electron spin resonance (ESR) method based on Cu2+ sensor. Cu2+ is reduced to Cu+ by the antioxidants in the fruits, and the remaining Cu2+ was determined by ESR and UV-Vis spectroscopy. Cu2+ can give an ESR signal whereas Cu+ cannot, and the loss of the ESR signal was used to quantify the antioxidant capacity of various fruits. The results were shown as vitamin C equivalent antioxidant capacity (VCEAC). The VCEAC values obtained by ESR and UV-Vis methods ranged from 24.23 to 688.61 mg/100 g and from 24.12 to 677.79 mg/100 g, respectively. Cupric ion reducing antioxidant capacity (CUPRAC) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) methods were employed for comparison. Based on Pearson's correlation test, the results obtained by CUPRAC and DPPH methods were both significantly correlated with these obtained by the present method, which indicated that the novel method was reliable. Total phenolic content for all kinds of fruits was measured with the Folin-Ciocalteu reagent, and VCEAC values obtained by the ESR method were significantly correlated with total phenolic contents. Graphical abstract.


Assuntos
Antioxidantes/análise , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Frutas/química , Fenóis/análise , Ácido Ascórbico/análise , Cátions Bivalentes/química , Cobre/química , Limite de Detecção , Oxirredução
20.
Proc Natl Acad Sci U S A ; 116(36): 17809-17816, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31383767

RESUMO

HIV type I (HIV-1) reverse transcriptase (RT) catalyzes the conversion of viral RNA into DNA, initiating the chain of events leading to integration of proviral DNA into the host genome. RT is expressed as a single polypeptide chain within the Gag-Pol polyprotein, and either prior to or following excision by HIV-1 protease forms a 66 kDa chain (p66) homodimer precursor. Further proteolytic attack by HIV-1 protease cleaves the ribonuclease H (RNase H) domain of a single subunit to yield the mature p66/p51 heterodimer. Here, we probe the spatial domain organization within the p66 homodimer using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure a large number of intra- and intersubunit distances between spin labels attached to surface-engineered cysteines. The DEER-derived distances are fully consistent with the structural subunit asymmetry found in the mature p66/p51 heterodimer in which catalytic activity resides in the p66 subunit, while the p51 subunit purely serves as a structural scaffold. Furthermore, the p66 homodimer precursor undergoes a conformational change involving the thumb, palm, and finger domains in one of the subunits (corresponding to the p66 subunit in the mature p66/p51 heterodimer) from a closed to a partially open state upon addition of a nonnucleoside inhibitor. The relative orientation of the domains was modeled by simulated annealing driven by the DEER-derived distances. Finally, the RNase H domain that is cleaved to generate p51 in the mature p66/p51 heterodimer is present in 2 major conformers. One conformer is fully solvent accessible thereby accounting for the observation that only a single subunit of the p66 homodimer precursor is susceptible to HIV-1 protease.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Transcriptase Reversa do HIV/química , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Transcriptase Reversa do HIV/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Coloração e Rotulagem , Relação Estrutura-Atividade
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