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1.
Nat Protoc ; 16(2): 893-918, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33442051

RESUMO

For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional 13C/15N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton (1H) detection schemes. We estimate that the entire preparative procedure until NMR experiments can be started takes 3-5 days.


Assuntos
Marcação por Isótopo/métodos , Espectroscopia de Ressonância Magnética/métodos , Proteínas/fisiologia , Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/metabolismo , Prótons
2.
ACS Appl Polym Mater ; 2(2): 515-527, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32090201

RESUMO

For realization of a wearable artificial kidney based on regeneration of a small volume of dialysate, efficient urea removal from dialysate is a major challenge. Here a potentially suitable polymeric sorbent based on phenylglyoxaldehyde (PGA), able to covalently bind urea under physiological conditions, is described. Sorbent beads containing PGA groups were obtained by suspension polymerization of either styrene or vinylphenylethan-1-one (VPE), followed by modification of the aromatic groups of poly(styrene) and poly(VPE) into PGA. It was found that PGA-functionalized sorbent beads had maximum urea binding capacities of 1.4-2.2 mmol/g and removed ∼0.6 mmol urea/g in 8 h at 37 °C under static conditions from urea-enriched phosphate-buffered saline, conditions representative of dialysate regeneration. This means that the daily urea production of a dialysis patient can be removed with a few hundred grams of this sorbent which, is an important step forward in the development of a wearable artificial kidney.

3.
Macromol Biosci ; 20(3): e1900396, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32065727

RESUMO

The aim of this study is to develop polymeric chemisorbents with a high density of ninhydrin groups, able to covalently bind urea under physiological conditions and thus potentially suitable for use in a wearable artificial kidney. Macroporous beads are prepared by suspension polymerization of 5-vinyl-1-indanone (vinylindanone) using a 90:10 (v/v) mixture of toluene and nitrobenzene as a porogen. The indanone groups are subsequently oxidized in a one-step procedure into ninhydrin groups. Their urea absorption kinetics are evaluated under both static and dynamic conditions at 37 °C in simulated dialysate (urea in phosphate buffered saline). Under static conditions and at a 1:1 molar ratio of ninhydrin: urea the sorbent beads remove ≈0.6-0.7 mmol g-1 and under dynamic conditions and at a 2:1 molar excess of ninhydrin ≈0.6 mmol urea g-1 sorbent in 8 h at 37 °C, which is a step toward a wearable artificial kidney.


Assuntos
Rins Artificiais , Ninidrina/química , Ureia/química , Dispositivos Eletrônicos Vestíveis , Adsorção , Humanos
4.
Angew Chem Int Ed Engl ; 58(37): 12969-12973, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31233270

RESUMO

Elucidating at atomic level how proteins interact and are chemically modified in cells represents a leading frontier in structural biology. We have developed a tailored solid-state NMR spectroscopic approach that allows studying protein structure inside human cells at atomic level under high-sensitivity dynamic nuclear polarization (DNP) conditions. We demonstrate the method using ubiquitin (Ub), which is critically involved in cellular functioning. Our results pave the way for structural studies of larger proteins or protein complexes inside human cells, which have remained elusive to in-cell solution-state NMR spectroscopy due to molecular size limitations.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Sequência de Aminoácidos , Células HeLa , Humanos , Modelos Moleculares , Conformação Proteica , Ubiquitina/química , Ubiquitinação
5.
Chem Commun (Camb) ; 53(28): 3933-3936, 2017 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-28327736

RESUMO

A combination of solid-state NMR techniques supported by EPR and SEM-EDX experiments was used to localize different carbon species (coke) in commercial fluid catalytic cracking catalysts. Aliphatic coke species formed during the catalytic process and aromatic coke species deposited directly from the feedstock respond differently to dynamic nuclear polarization signal enhancement in integral and crushed FCC particles, indicating that aromatic species are mostly concentrated on the outside of the catalyst particles, whereas aliphatic species are also located on the inside of the FCC particles. The comparison of solid-state NMR data with and without the DNP radical at low and ambient temperature suggests the proximity between aromatic carbon deposits and metals (mostly iron) on the catalyst surface. These findings potentially indicate that coke and iron deposit together, or that iron has a role in the formation of aromatic coke.

6.
J Biomol NMR ; 60(2-3): 157-68, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25284462

RESUMO

Dynamic nuclear polarization (DNP) has become a powerful method to enhance spectroscopic sensitivity in the context of magnetic resonance imaging and nuclear magnetic resonance spectroscopy. We show that, compared to DNP at lower field (400 MHz/263 GHz), high field DNP (800 MHz/527 GHz) can significantly enhance spectral resolution and allows exploitation of the paramagnetic relaxation properties of DNP polarizing agents as direct structural probes under magic angle spinning conditions. Applied to a membrane-embedded K(+) channel, this approach allowed us to refine the membrane-embedded channel structure and revealed conformational substates that are present during two different stages of the channel gating cycle. High-field DNP thus offers atomic insight into the role of molecular plasticity during the course of biomolecular function in a complex cellular environment.


Assuntos
Proteínas de Bactérias/química , Campos Magnéticos , Ressonância Magnética Nuclear Biomolecular , Canais de Potássio/química , Isótopos de Carbono , Ativação do Canal Iônico , Solventes , Temperatura
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