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1.
Anal Chem ; 93(8): 3976-3986, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33577736

RESUMO

We have applied nuclear magnetic resonance spectroscopy based plasma phenotyping to reveal diagnostic molecular signatures of SARS-CoV-2 infection via combined diffusional and relaxation editing (DIRE). We compared plasma from healthy age-matched controls (n = 26) with SARS-CoV-2 negative non-hospitalized respiratory patients and hospitalized respiratory patients (n = 23 and 11 respectively) with SARS-CoV-2 rRT-PCR positive respiratory patients (n = 17, with longitudinal sampling time-points). DIRE data were modelled using principal component analysis and orthogonal projections to latent structures discriminant analysis (O-PLS-DA), with statistical cross-validation indices indicating excellent model generalization for the classification of SARS-CoV-2 positivity for all comparator groups (area under the receiver operator characteristic curve = 1). DIRE spectra show biomarker signal combinations conferred by differential concentrations of metabolites with selected molecular mobility properties. These comprise the following: (a) composite N-acetyl signals from α-1-acid glycoprotein and other glycoproteins (designated GlycA and GlycB) that were elevated in SARS-CoV-2 positive patients [p = 2.52 × 10-10 (GlycA) and 1.25 × 10-9 (GlycB) vs controls], (b) two diagnostic supramolecular phospholipid composite signals that were identified (SPC-A and SPC-B) from the -+N-(CH3)3 choline headgroups of lysophosphatidylcholines carried on plasma glycoproteins and from phospholipids in high-density lipoprotein subfractions (SPC-A) together with a phospholipid component of low-density lipoprotein (SPC-B). The integrals of the summed SPC signals (SPCtotal) were reduced in SARS-CoV-2 positive patients relative to both controls (p = 1.40 × 10-7) and SARS-CoV-2 negative patients (p = 4.52 × 10-8) but were not significantly different between controls and SARS-CoV-2 negative patients. The identity of the SPC signal components was determined using one and two dimensional diffusional, relaxation, and statistical spectroscopic experiments. The SPCtotal/GlycA ratios were also significantly different for control versus SARS-CoV-2 positive patients (p = 1.23 × 10-10) and for SARS-CoV-2 negatives versus positives (p = 1.60 × 10-9). Thus, plasma SPCtotal and SPCtotal/GlycA are proposed as sensitive molecular markers for SARS-CoV-2 positivity that could effectively augment current COVID-19 diagnostics and may have value in functional assessment of the disease recovery process in patients with long-term symptoms.


Assuntos
/diagnóstico , Orosomucoide/análise , Fosfolipídeos/sangue , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Ressonância Magnética Nuclear Biomolecular/métodos , Orosomucoide/química , Fosfolipídeos/química , Espectroscopia de Prótons por Ressonância Magnética/métodos , Espectroscopia de Prótons por Ressonância Magnética/estatística & dados numéricos , Curva ROC
2.
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
3.
Nat Commun ; 12(1): 691, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514730

RESUMO

Methyl-specific isotope labeling is a powerful tool to study the structure, dynamics and interactions of large proteins and protein complexes by solution-state NMR. However, widespread applications of this methodology have been limited by challenges in obtaining confident resonance assignments. Here, we present Methyl Assignments Using Satisfiability (MAUS), leveraging Nuclear Overhauser Effect cross-peak data, peak residue type classification and a known 3D structure or structural model to provide robust resonance assignments consistent with all the experimental inputs. Using data recorded for targets with known assignments in the 10-45 kDa size range, MAUS outperforms existing methods by up to 25,000 times in speed while maintaining 100% accuracy. We derive de novo assignments for multiple Cas9 nuclease domains, demonstrating that the methyl resonances of multi-domain proteins can be assigned accurately in a matter of days, while reducing biases introduced by manual pre-processing of the raw NOE data. MAUS is available through an online web-server.


Assuntos
Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Algoritmos , Proteína 9 Associada à CRISPR/ultraestrutura , Isótopos de Carbono , Interleucina-2/química , Interleucina-2/isolamento & purificação , Marcação por Isótopo/métodos , Ressonância Magnética Nuclear Biomolecular/instrumentação , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Streptococcus pyogenes/enzimologia , Trítio
4.
Methods Mol Biol ; 2167: 183-202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712921

RESUMO

A full understanding of biomolecular function requires an analysis of both the dynamic properties of the system of interest and the identification of those dynamics that are required for function. We describe NMR methods based on metabolically directed specific isotope labeling for the identification of molecular disorder and/or conformational transitions on the RNA backbone ribose groups. These analyses are complemented by the use of synthetic covalently modified nucleotides constrained to a single sugar pucker, which allow functional assessment of dynamics by selectively removing a minor conformer identified by NMR from the structural ensemble.


Assuntos
Ensaios Enzimáticos/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/genética , Ribose/química , Isótopos de Carbono/química , Cinética , Nucleotídeos , Relação Quantitativa Estrutura-Atividade , RNA não Traduzido/química , RNA não Traduzido/genética
5.
Nat Commun ; 11(1): 5317, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087707

RESUMO

Multidimensional TOCSY and NOESY are central experiments in chemical and biophysical NMR. Limited efficiencies are an intrinsic downside of these methods, particularly when targeting labile sites. This study demonstrates that the decoherence imparted on these protons through solvent exchanges can, when suitably manipulated, lead to dramatic sensitivity gains per unit time in the acquisition of these experiments. To achieve this, a priori selected frequencies are encoded according to Hadamard recipes, while concurrently subject to looped selective inversion or selective saturation procedures. Suitable processing then leads to protein, oligosaccharide and nucleic acid cross-peak enhancements of ≈200-1000% per scan, in measurements that are ≈10-fold faster than conventional counterparts. The extent of these gains will depend on the solvent exchange and relaxation rates of the targeted sites; these gains also benefit considerably from the spectral resolution provided by ultrahigh fields, as corroborated by NMR experiments at 600 MHz and 1 GHz. The mechanisms underlying these experiments' enhanced efficiencies are analyzed on the basis of three-way polarization transfer interplays between the water, labile and non-labile protons, and the experimental results are rationalized using both analytical and numerical derivations. Limitations as well as further extensions of the proposed methods, are also discussed.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Ácidos Nucleicos/química , Polissacarídeos/química , Proteínas/química , Sítios de Ligação , Espectroscopia de Ressonância Magnética/estatística & dados numéricos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/métodos
6.
Nat Protoc ; 15(10): 3527-3555, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968252

RESUMO

Solid acid catalysts are used extensively in various advanced chemical and petrochemical processes. Their catalytic performance (namely, activity, selectivity, and reaction pathway) mostly depends on their acid properties, such as type (Brønsted versus Lewis), location, concentration, and strength, as well as the spatial correlations of their acid sites. Among the diverse methods available for acidity characterization, solid-state nuclear magnetic resonance (SSNMR) techniques have been recognized as the most valuable and reliable tool, especially in conjunction with suitable probe molecules that possess observable nuclei with desirable properties. Taking 31P probe molecules as an example, both trimethylphosphine (TMP) and trimethylphosphine oxide (TMPO) adsorb preferentially to the acid sites on solid catalysts and thus are capable of providing qualitative and quantitative information for both Brønsted and Lewis acid sites. This protocol describes procedures for (i) the pretreatment of typical solid acid catalysts, (ii) adoption and adsorption of various 31P probe molecules, (iii) considerations for one- and two-dimensional (1D and 2D, respectively) NMR acquisition, (iv) relevant data analysis and spectral assignment, and (v) methodology for NMR mapping with the assistance of theoretical calculations. Users familiar with SSNMR experiments can complete 31P-1H heteronuclear correlation (HETCOR), 31P-31P proton-driven spin diffusion (PDSD), and double-quantum (DQ) homonuclear correlation with this protocol within 2-3 d, depending on the complexity and the accessible acid sites of the solid acid samples.


Assuntos
Ácidos/química , Catálise , Ressonância Magnética Nuclear Biomolecular/métodos , Imagem por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Fosfinas/química , Prótons
7.
Proc Natl Acad Sci U S A ; 117(35): 21014-21021, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817429

RESUMO

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Imagem por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Permeabilidade , Estrutura Secundária de Proteína
8.
Proc Natl Acad Sci U S A ; 117(23): 12836-12846, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457157

RESUMO

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)-based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics. We illustrate the methodology with studies of the 153-base pair Widom DNA molecule that is simultaneously methyl-labeled at five sites, showing that high-quality 13C-1H spectra can be recorded on 100 µM samples in a few minutes. NMR spin relaxation studies of labeled methyl groups in both DNA and the H2B histone protein component of the 200-kDa nucleosome core particle (NCP) establish that methyl groups at 5mC and 6mA positions are, in general, more rigid than Ile, Leu, and Val methyl probes in protein side chains. Studies focusing on histone H2B of NCPs wrapped with either wild-type DNA or DNA methylated at all 26 CpG sites highlight the utility of NMR in investigating the structural dynamics of the NCP and how its histone core is affected through DNA methylation, an important regulator of transcription.


Assuntos
DNA/ultraestrutura , Ressonância Magnética Nuclear Biomolecular/métodos , Nucleossomos/ultraestrutura , Análise Espectral/métodos , Adenina/química , Isótopos de Carbono , Ilhas de CpG , Citosina/química , DNA/química , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Simulação de Dinâmica Molecular , Peso Molecular
9.
Nucleic Acids Res ; 48(W1): W36-W40, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32459331

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy data provides valuable information on the behaviour of proteins in solution. The primary data to determine when studying proteins are the per-atom NMR chemical shifts, which reflect the local environment of atoms and provide insights into amino acid residue dynamics and conformation. Within an amino acid residue, chemical shifts present multi-dimensional and complexly cross-correlated information, making them difficult to analyse. The ShiftCrypt method, based on neural network auto-encoder architecture, compresses the per-amino acid chemical shift information in a single, interpretable, amino acid-type independent value that reflects the biophysical state of a residue. We here present the ShiftCrypt web server, which makes the method readily available. The server accepts chemical shifts input files in the NMR Exchange Format (NEF) or NMR-STAR format, executes ShiftCrypt and visualises the results, which are also accessible via an API. It also enables the "biophysically-based" pairwise alignment of two proteins based on their ShiftCrypt values. This approach uses Dynamic Time Warping and can optionally include their amino acid code information, and has applications in, for example, the alignment of disordered regions. The server uses a token-based system to ensure the anonymity of the users and results. The web server is available at www.bio2byte.be/shiftcrypt.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Software , Aminoácidos/química , Redes Neurais de Computação , Desnaturação Proteica , Dobramento de Proteína , Desdobramento de Proteína
10.
J Med Chem ; 63(10): 5367-5386, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32342688

RESUMO

In search of novel drugs against tuberculosis, we previously discovered and profiled a novel hydantoin-based family that demonstrated highly promising in vitro potency against Mycobacterium. tuberculosis. The compounds were found to be noncovalent inhibitors of DprE1, a subunit of decaprenylphosphoryl-ß-d-ribose-2'-epimerase. This protein, localized in the periplasmic space of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug target. Here, we report the further SAR exploration of this chemical family through more than 80 new analogues. Among these, the most active representatives combined submicromolar cellular potency and nanomolar target affinity with balanced physicochemical properties and low human cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family of DprE1 inhibitors represents a promising noncovalent lead series for the discovery of novel antituberculosis agents.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Hidantoínas/química , Hidantoínas/farmacologia , Oxirredutases do Álcool/metabolismo , Animais , Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Feminino , Células Hep G2 , Humanos , Hidantoínas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo
11.
PLoS One ; 15(4): e0232015, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330166

RESUMO

Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.


Assuntos
Conformação Proteica , Proteínas/metabolismo , Ribossomos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica/fisiologia , Estabilidade Proteica , Ribonuclease Pancreático/metabolismo
12.
Chemistry ; 26(30): 6789-6792, 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32240561

RESUMO

Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time 31 P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.


Assuntos
Diacilglicerol Quinase/metabolismo , Escherichia coli/metabolismo , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/metabolismo , Catálise , Fenômenos Fisiológicos Celulares , Diacilglicerol Quinase/química , Escherichia coli/química , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosforilação
13.
Int J Mol Sci ; 21(5)2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32155847

RESUMO

Solution NMR spectroscopy is a unique and powerful technique that has the ability to directly connect the structural dynamics of proteins in physiological conditions to their activity and function. Here, we summarize recent studies in which solution NMR contributed to the discovery of relationships between key dynamic properties of proteins and functional mechanisms in important biological systems. The capacity of NMR to quantify the dynamics of proteins over a range of time scales and to detect lowly populated protein conformations plays a critical role in its power to unveil functional protein dynamics. This analysis of dynamics is not only important for the understanding of biological function, but also in the design of specific ligands for pharmacologically important proteins. Thus, the dynamic view of structure provided by NMR is of importance in both basic and applied biology.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas/metabolismo , Animais , Humanos , Conformação Proteica
14.
J Mol Biol ; 432(9): 2949-2972, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32135192

RESUMO

Proteins and their interactions control a plethora of biological functions and enable life. Protein-protein interactions can be highly dynamic, involve proteins with different degrees of "foldedness," and are often regulated through an intricate network of post-translational modifications. Central parts of protein-protein networks are intrinsically disordered proteins (IDPs). IDPs act as regulatory interaction hubs, enabled by their flexible nature. They employ various modes of binding mechanisms, from folding upon ligand binding to formation of highly dynamic "fuzzy" protein-protein complexes. Mutations or perturbations in regulation of IDPs are hallmarks of many diseases. Protein surfaces play key roles in protein-protein interactions. However, protein surfaces and protein surface accessibility are difficult to study experimentally. NMR-based solvent paramagnetic relaxation enhancement (sPRE) can provide quantitative experimental information on protein surface accessibility, which can be further used to obtain distance information for structure determination, identification of interaction surfaces, conformational changes, and identification of low-populated transient structure and long-range contacts in IDPs and dynamic protein-protein interactions. In this review, we present and discuss state-of the art sPRE techniques and their applications to investigate structure and dynamics of IDPs and protein-protein interactions. Finally, we provide an outline for potential future applications of the sPRE approach in combination with complementary techniques and modeling, to study novel paradigms, such as liquid-liquid phase separation, regulation of IDPs and protein-protein interactions by post-translational modifications, and targeting of disordered proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Solventes/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional
15.
Chem Commun (Camb) ; 56(19): 2897-2900, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32037418

RESUMO

[Tm(DPA)3]3- was used to generate multiple, paramagnetic nuclear Overhauser effect NMR spectra of cationic peptides when weakly bound to a lipopolysaccharide micelle. Increased spectral resolution combined with a marked increase in the number of distance restraints yielded high resolution structures of polymyxin and MSI-594 in the liposaccharide bound state.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Elementos da Série dos Lantanídeos/química , Micelas , Ressonância Magnética Nuclear Biomolecular/métodos , Indicadores e Reagentes/química , Peptídeos/química , Polimixina B/química , Conformação Proteica
16.
J Mol Biol ; 432(9): 2913-2929, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32044345

RESUMO

Nuclear magnetic resonance (NMR) structure refinement is inherently integrative. The refinement incorporates a multitude of experimental data and minimizes the difference between observed and calculated values. Here, we review how the integrative use of non-NMR measurements, in particular, distance restraints from Förster resonance energy transfer and cross-linking coupled with mass spectrometry, can augment NMR depiction of protein structure and dynamics. Refinement against both NMR and non-NMR distance restraints helps to characterize the structures of high-molecular-weight proteins and protein complexes. When a protein fluctuates among multiple conformations at millisecond or a faster timescale, NMR signals from the different conformational states may coalesce into a single set of peaks. The integration of non-NMR distance restraints facilitates the deconvolution of NMR observables to state-specific restraints. Furthermore, the integrative use of fluorescence measurements, which provides an assessment of both length scale and timescale of protein dynamics simplifies protein ensemble structure refinement otherwise with NMR restraints alone and affords a more wholesome picture of protein dynamics. Together, distance measurements are intuitive and easy to implement by using an appropriate pseudoenergy function. Future development shall involve more accurate modeling of paramagnetic and fluorescent probes, incorporation of sparse restraints from new techniques, and characterization of protein structures in a complex cellular environment.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Algoritmos , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Conformação Proteica
17.
Anal Chim Acta ; 1104: 105-109, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32106940

RESUMO

Solid-state 13C and 19F NMR spectroscopy offers a non-destructive, highly selective protocol for the identification of forensically relevant synthetic cannabinoids on herbal substrates. Using this technique, well resolved 13C spectra were obtained that readily enabled structural identification; in some instances complemented by 19F spectral data. The approach described has potential for related applications such as the direct detection of pesticides on plants.


Assuntos
Canabinoides/análise , Drogas Desenhadas/análise , Ressonância Magnética Nuclear Biomolecular/métodos , Preparações de Plantas/química , Turnera/química , Canabinoides/química , Drogas Desenhadas/química , Estrutura Molecular
18.
Biochemistry ; 59(6): 755-765, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31909602

RESUMO

Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologues, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show the conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogues. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5'-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B2 subsite ligand preferences, our results show a lack of typical B2 subsite interactions normally observed in homologous ligand-bound RNases. A comparison of the dynamical properties of RNase 6 in its apo-, substrate-, and product-bound states highlight the unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ribonucleases/química , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação/fisiologia , Humanos , Ligantes , Estrutura Secundária de Proteína
19.
Biochemistry ; 59(6): 733-735, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31975578

RESUMO

We assessed the ability of two strains of Escherichia coli, BL21 (DE3) and Tuner (DE3), to express a variant of the B1 domain of protein G, which forms a side-by-side dimer, by using fluorine-labeling and 19F nuclear magnetic resonance spectroscopy. BL21 cells express the protein in a binary, all-or-none, manner, where more cells express the protein at a high level with an increasing inducer concentration. Tuner cells express the protein in a rheostatic manner, where expression increases across all cells with an increasing inducer concentration.


Assuntos
Proteínas de Escherichia coli/biossíntese , Ressonância Magnética Nuclear Biomolecular/métodos , Proteômica/métodos , Proteínas Recombinantes/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Expressão Gênica , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/genética
20.
J Leukoc Biol ; 107(6): 1115-1122, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31965639

RESUMO

Chemokines are small soluble proteins that drive cell migration through the formation of concentration gradients. Chemokine binding to G protein-coupled chemokine receptors in the cell membrane activates intracellular signaling pathways and is a fundamental process involved in numerous physiological and pathophysiological functions. In the past few years, significant experimental developments have made it possible to characterize complexes between chemokine receptors and chemokines at a molecular level. Here, I review these developments from an experimental perspective, focusing on how the ability to express, purify, and stabilize receptor:chemokine complexes have made studies by X-ray crystallography, nuclear magnetic resonance, and other methods possible. I give examples of how these studies have advanced our understanding of the architecture of receptor:chemokine complexes as well as the mechanisms involved in complex formation. Finally, I discuss some of the many remaining questions and challenges that will require studies of more receptors and chemokines as well as further development of experimental methods.


Assuntos
Quimiocinas/química , Glicosaminoglicanos/química , Receptores de Quimiocinas/química , Sítios de Ligação , Quimiocinas/genética , Quimiocinas/metabolismo , Cristalografia por Raios X/métodos , Expressão Gênica , Glicosaminoglicanos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais
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