RESUMO
We have developed technology for producing accurate spectral fingerprints of small molecules through modeling of NMR spin system matrices to encapsulate their chemical shifts and scalar couplings. We describe here how libraries of these spin systems utilizing unique and reproducible atom numbering can be used to improve NMR-based ligand screening and metabolomics studies. We introduce new Web services that facilitate the analysis of NMR spectra of mixtures of small molecules to yield their identification and quantification. The library of parametrized compounds has been expanded to cover simulations of 1H NMR spectra at a variety of magnetic fields of more than 1100 compounds, included are many common metabolites and a library of drug-like molecular fragments used in ligand screening. The compound library and related Web services are freely available from http://gissmo.nmrfam.wisc.edu/ .
RESUMO
Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.
Assuntos
Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/isolamento & purificação , Arabidopsis/genética , Biossíntese de Proteínas , Proteínas RGS/biossíntese , Proteínas RGS/isolamento & purificação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Proteínas RGS/química , Proteínas RGS/genéticaRESUMO
Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.
Assuntos
Sistema Livre de Células , Biossíntese de Proteínas/genética , Proteínas/genética , Clonagem Molecular , Escherichia coli/genética , Eucariotos/genética , Expressão Gênica , Vetores Genéticos , Células Germinativas , Proteínas/isolamento & purificação , Triticum/genéticaRESUMO
Flagellin-sensing 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A), while the acidic phosphomimic mutants FLS2(S938D) and FLS2(S938E) conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A), demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A) and FLS2(S938D) mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A) or FLS2(D997A) (a kinase catalytic site mutant), but was normally induced in FLS2(S938D) plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Flagelina/imunologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Flagelina/metabolismo , Regulação da Expressão Gênica de Plantas , Fosforilação , Plantas Geneticamente Modificadas , Proteínas Quinases/química , Proteínas Quinases/genética , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de SinaisRESUMO
The Ebola Virus is a causative agent of viral hemorrhagic fever outbreaks and a potential global health risk. The outbreak in West Africa (2013-2016) led to 11,000+ deaths and 30,000+ Ebola infected individuals. The current outbreak in the Democratic Republic of Congo (DRC) with 3000+ confirmed cases and 2000+ deaths attributed to Ebola virus infections provides a reminder that innovative countermeasures are still needed. Ebola virus encodes 7 open reading frames (ORFs). Of these, the nucleocapsid protein (eNP) encoded by the first ORF plays many significant roles, including a role in viral RNA synthesis. Here we describe efforts to target the C-terminal domain of eNP (eNP-CTD) that contains highly conserved residues 641-739 as a pan-Ebola antiviral target. Interactions of eNP-CTD with Ebola Viral Protein 30 (eVP30) and Viral Protein 40 (eVP40) have been shown to be crucial for viral RNA synthesis, virion formation, and virion transport. We used nuclear magnetic response (NMR)-based methods to screened the eNP-CTD against a fragment library. Perturbations of 1D 1H NMR spectra identified of 48 of the 439 compounds screened as potential eNP CTD interactors. Subsequent analysis of these compounds to measure chemical shift perturbations in 2D 1H,15N NMR spectra of 15N-labeled protein identified six with low millimolar affinities. All six perturbed an area consisting mainly of residues at or near the extreme C-terminus that we named "Site 1" while three other sites were perturbed by other compounds. Our findings here demonstrate the potential utility of eNP as a target, several fragment hits, and provide an experimental pipeline to validate viral-viral interactions as potential panfiloviral inhibitor targets.
Assuntos
Ebolavirus/química , Nucleoproteínas/química , Relação Estrutura-Atividade , Descoberta de Drogas , Ebolavirus/genética , Biblioteca Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Nucleoproteínas/genética , Replicação ViralRESUMO
The Center for Eukaryotic Structural Genomics (CESG) is a "specialized" or "technology development" center supported by the Protein Structure Initiative (PSI). CESG's mission is to develop improved methods for the high-throughput solution of structures from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes. Notably, 30% of all structures of human proteins solved by the PSI Centers were determined at CESG. Whereas eukaryotic proteins generally are considered to be much more challenging targets than prokaryotic proteins, the technology now in place at CESG yields success rates that are comparable to those of the large production centers that work primarily on prokaryotic proteins. We describe here the technological innovations that underlie CESG's platforms for bioinformatics and laboratory information management, target selection, protein production, and structure determination by X-ray crystallography or NMR spectroscopy.
Assuntos
Genômica/organização & administração , Proteínas/química , Animais , Cristalografia por Raios X , Genômica/métodos , Humanos , Sistemas Multi-Institucionais/organização & administração , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas/genética , Proteômica/organização & administraçãoRESUMO
Ebola viral infections have resulted in several deadly epidemics in recent years in West and Central Africa. Because only one of the seven proteins encoded by the viral genome possesses enzymatic activity, disruption of protein-protein interactions is a promising route for antiviral drug development. We carried out a screening campaign to identify small, drug-like compounds that bind to the C-terminal region of the multifunctional Ebola nucleoprotein (eNP) with the objective of discovering ones that disrupt its binding to other Ebola proteins or to the single-stranded RNA genome. In the course of this effort we assigned the backbone 1H, 15N, and 13C resonances of residues 600â739, the region that contains the critical eVP30 binding region 600â615 targeted by host factors, and used the assigned chemical shifts to predict secondary structural features and peptide dynamics. This work supports and extends the previous X-ray crystal structures and NMR studies of residues 641â739. We found that the 600â739 domain consists of separate regions that are largely disordered and ordered.
Assuntos
Ebolavirus , Ressonância Magnética Nuclear Biomolecular , Nucleoproteínas/química , Proteínas Virais/química , Estrutura Secundária de ProteínaRESUMO
The crystal structure of the protein product of the gene locus At1g05000, a hypothetical protein from A. thaliana, was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 20.4% (R(free) = 24.9%) at 3.3 A. The protein adopts the alpha/beta fold found in cysteine phosphatases, a superfamily of phosphatases that possess a catalytic cysteine and form a covalent thiol-phosphate intermediate during the catalytic cycle. In At1g05000, the analogous cysteine (Cys(150)) is located at the bottom of a positively-charged pocket formed by residues that include the conserved arginine (Arg(156)) of the signature active site motif, HCxxGxxRT. Of 74 model phosphatase substrates tested, purified recombinant At1g05000 showed highest activity toward polyphosphate (poly-P(12-13)) and deoxyribo- and ribonucleoside triphosphates, and less activity toward phosphoenolpyruvate, phosphotyrosine, phosphotyrosine-containing peptides, and phosphatidyl inositols. Divalent metal cations were not required for activity and had little effect on the reaction.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Domínio Catalítico , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Monoéster Fosfórico Hidrolases/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genoma de Planta , Proteínas de Arabidopsis/isolamento & purificação , Sistema Livre de Células , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Sementes/genética , Triticum/genéticaRESUMO
The crystal structure of the gene product of At3g21360 from Arabidopsis thaliana was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 19.3% (Rfree = 24.1%) at 2.4 A resolution. The crystal structure includes two monomers in the asymmetric unit that differ in the conformation of a flexible domain that spans residues 178-230. The crystal structure confirmed that At3g21360 encodes a protein belonging to the clavaminate synthase-like superfamily of iron(II) and 2-oxoglutarate-dependent enzymes. The metal-binding site was defined and is similar to the iron(II) binding sites found in other members of the superfamily.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Proteínas Tirosina Fosfatases/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Fosfatases de Especificidade Dupla , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29-33 and 39-43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.
Assuntos
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Escherichia coli/metabolismo , Frutas , Genes de Plantas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria , Paladar , Raios Ultravioleta , Água/químicaRESUMO
Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.
Assuntos
Cisteína Endopeptidases/química , Rhinovirus/enzimologia , Proteínas Virais/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Sugar methyltransferases (MTs) are an important class of tailoring enzymes that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to sugar-based N-, C- and O-nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using (1)H-(13)C-gHSQC with isotopically labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3'-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal- and general acid/base-dependent O-methyltransferase, and as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering.
Assuntos
Aminoglicosídeos/biossíntese , Metiltransferases/química , Metiltransferases/metabolismo , Ramnose/química , Catálise , Domínio Catalítico , Enedi-Inos , Espectroscopia de Ressonância Magnética , Micromonospora/enzimologia , Modelos Moleculares , Estrutura MolecularAssuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografia por Raios X , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Marcação por Isótopo/métodos , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.
Assuntos
Células Eucarióticas/química , Proteínas/isolamento & purificação , Sequência de Aminoácidos , Animais , Automação , Sequência de Bases , Cromatografia de Afinidade , Cristalização , Eletroforese em Gel de Ágar/métodos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/isolamento & purificação , Humanos , Camundongos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Plasmídeos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Xenopus laevisRESUMO
Cysteine dioxygenase (CDO) catalyzes the oxidation of l-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Angstroms. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical beta-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.