RESUMEN
The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.
Asunto(s)
Antibacterianos/farmacología , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas/efectos de los fármacos , Animales , Antibacterianos/química , Compuestos Aza/química , Compuestos Aza/farmacología , Ciclooctanos/química , Ciclooctanos/farmacología , Femenino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , beta-LactamasasRESUMEN
Disruption of the HBV capsid assembly process through small-molecule interaction with HBV core protein is a validated target for the suppression of hepatitis B viral replication and the development of new antivirals. Through combination of key structural features associated with two distinct series of capsid assembly modulators, a novel aminochroman-based chemotype was identified. Optimization of anti-HBV potency through generation of SAR in addition to further core modifications provided a series of related functionalized aminoindanes. Key compounds demonstrated excellent cellular potency in addition to favorable ADME and pharmacokinetic profiles and were shown to be highly efficacious in a mouse model of HBV replication. Aminoindane derivative AB-506 was subsequently advanced into clinical development.
Asunto(s)
Antivirales , Proteínas de la Cápside , Cápside , Animales , Ratones , Antivirales/farmacología , Modelos Animales de Enfermedad , Relación Estructura-Actividad , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/metabolismoRESUMEN
Double electron-electron resonance in conjunction with site-directed spin labeling has been used to probe natural conformational sampling of the human tumor necrosis factor α trimer. We suggest a previously unreported, predeoligomerization conformation of the trimer that has been shown to be sampled at low frequency. A model of this trimeric state has been constructed based on crystal structures using the double-electron-electron-resonance distances. The model shows one of the protomers to be rotated and tilted outward at the tip end, leading to a breaking of the trimerous symmetry and distortion at a receptor-binding interface. The new structure offers opportunities to modulate the biological activity of tumor necrosis factor α through stabilization of the distorted trimer with small molecules.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Factor de Necrosis Tumoral alfa/metabolismo , Escherichia coli , Humanos , Modelos Moleculares , Mutación , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Marcadores de Spin , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/metabolismo , Modelos Moleculares , Transactivadores/química , Regulación Alostérica/fisiología , Calorimetría , Clonación Molecular , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Oligonucleótidos/genética , Estructura Terciaria de Proteína , Análisis Espectral , Factores de Tiempo , Transactivadores/metabolismo , Regulador Transcripcional ERGRESUMEN
Epilepsy is a common neurological disorder characterized by abnormal activity of neuronal networks, leading to seizures. The racetam class of anti-seizure medications bind specifically to a membrane protein found in the synaptic vesicles of neurons called synaptic vesicle protein 2 (SV2) A (SV2A). SV2A belongs to an orphan subfamily of the solute carrier 22 organic ion transporter family that also includes SV2B and SV2C. The molecular basis for how anti-seizure medications act on SV2s remains unknown. Here we report cryo-electron microscopy structures of SV2A and SV2B captured in a luminal-occluded conformation complexed with anticonvulsant ligands. The conformation bound by anticonvulsants resembles an inhibited transporter with closed luminal and intracellular gates. Anticonvulsants bind to a highly conserved central site in SV2s. These structures provide blueprints for future drug design and will facilitate future investigations into the biological function of SV2s.
RESUMEN
In this work, electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography were used to examine the origins of EPR line shapes from spin-labels at the protein-lipid interface on the ß-barrel membrane protein BtuB. Two atomic-resolution structures were obtained for the methanethiosulfonate spin-label derivatized to cysteines on the membrane-facing surface of BtuB. At one of these sites, position 156, the label side chain resides in a pocket formed by neighboring residues; however, it extends from the protein surface and yields a single-component EPR spectrum in the crystal that results primarily from fast rotation about the fourth and fifth bonds linking the spin-label to the protein backbone. In lipid bilayers, site 156 yields a multicomponent spectrum resulting from different rotameric states of the labeled side chain. Moreover, changes in the lipid environment, such as variations in bilayer thickness, modulate the EPR spectrum by modulating label rotamer populations. At a second site, position 371, the labeled side chain interacts with a pocket on the protein surface, leading to a highly immobilized single-component EPR spectrum that is not sensitive to hydrocarbon thickness. This spectrum is similar to that seen at other sites that are deep in the hydrocarbon, such as position 170. This work indicates that the rotameric states of spin-labels on exposed hydrocarbon sites are sensitive to the environment at the protein-hydrocarbon interface, and that this environment may modulate weak interactions between the labeled side chain and the protein surface. In the case of BtuB, lipid acyl chain packing is not symmetric around the ß-barrel, and EPR spectra from labeled hydrocarbon-facing sites in BtuB may reflect this asymmetry. In addition to facilitating the interpretation of EPR spectra of membrane proteins, these results have important implications for the use of long-range distance restraints in protein structure refinement that are obtained from spin-labels.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Proteínas de Escherichia coli/química , Proteínas de Transporte de Membrana/química , Sitios de Unión , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Modelos Moleculares , Mutagénesis , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solventes , Marcadores de SpinRESUMEN
Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B(12). Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Secuencias de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutagénesis , Mutación , Conformación Proteica , Pliegue de Proteína , Desplegamiento Proteico , Marcadores de Spin , TermodinámicaRESUMEN
Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.
Asunto(s)
Sistemas de Transporte de Aminoácidos/química , Proteínas Bacterianas/química , Óxidos N-Cíclicos/química , Sistemas de Transporte de Aminoácidos/genética , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Estructura Secundaria de Proteína/efectos de los fármacos , Marcadores de SpinRESUMEN
Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.
Asunto(s)
Chlamydia trachomatis/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Neisseria gonorrhoeae/enzimología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Modelos Moleculares , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadAsunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacterias Gramnegativas/metabolismo , Transporte Biológico Activo , Membrana Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación ProteicaRESUMEN
Thiamine monophosphate kinase (ThiL) catalyzes the last step of thiamine pyrophosphate (TPP) synthesis, the ATP-dependent phosphorylation of thiamine monophosphate (TMP) to thiamine pyrophosphate. We solved the structure of ThiL from the human pathogen A. baumanii in complex with a pair of substrates TMP and a non-hydrolyzable adenosine triphosphate analog, and in complex with a pair of products TPP and adenosine diphosphate. High resolution of the data and anomalous diffraction allows for a detailed description of the binding mode of substrates and products, and their metal environment. The structures further support a previously proposed in-line attack reaction mechanism and show a distinct variability of metal content of the active site.
Asunto(s)
Acinetobacter baumannii/enzimología , Acinetobacter baumannii/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Cristalización , Tiamina Pirofosfato/metabolismoRESUMEN
Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.
Asunto(s)
Membrana Celular/enzimología , Proteínas de la Membrana/química , Metaloendopeptidasas/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin to form protoheme IX. The crystal structures of human ferrochelatase both with and without the protoporphyrin substrate bound have been determined previously. The substrate-free enzyme has an open active site pocket, while in the substrate-bound enzyme, the active site pocket is closed around the porphyrin macrocycle and a number of active site residues have reoriented side chains. To understand how and why these structural changes occur, we have substituted three amino acid residues (H263, H341, and F337) whose side chains occupy different spatial positions in the substrate-free versus substrate-bound ferrochelatases. The catalytic and structural properties of ferrochelatases containing the amino acid substitutions H263C, H341C, and F337A were examined. It was found that in the H263C and H341C variants, but not the F337A variant enzymes, the side chains of N75, M76, R164, H263, F337, H341, and E343 are oriented in a fashion similar to what is found in ferrochelatase with the bound porphyrin substrate. However, all of the variant forms possess open active site pockets which are found in the structure of porphyrin-free ferrochelatase. Thus, while the interior walls of the active site pocket are remodeled in these variants, the exterior lips remain unaltered in position. One possible explanation for this collective reorganization of active site side chains is the presence of a hydrogen bond network among H263, H341, and E343. This network is disrupted in the variants by alteration of H263C or H341C. In the substrate-bound enzyme, the formation of a hydrogen bond between H263 and a pyrrole nitrogen results in disruption of the network. The possible role of this network in catalysis is discussed.
Asunto(s)
Ferroquelatasa/metabolismo , Sitios de Unión , Catálisis , Ferroquelatasa/química , Humanos , Enlace de Hidrógeno , Modelos MolecularesRESUMEN
Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.
Asunto(s)
Proteínas Bacterianas/química , Clostridium thermocellum/metabolismo , Escherichia coli/metabolismo , Shewanella/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Clonación Molecular , ADN/química , Cartilla de ADN/química , Vectores Genéticos , Calor , Proteínas de Unión a Maltosa , Modelos Estadísticos , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Reacción en Cadena de la Polimerasa , Potyvirus/metabolismo , Proteoma , Proteómica/métodos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes/química , Recombinación Genética , Solubilidad , TemperaturaRESUMEN
Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Cristalización , Proteínas/aislamiento & purificaciónRESUMEN
Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2,200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.
Asunto(s)
Proteínas Arqueales/aislamiento & purificación , Expresión Génica , Modelos Moleculares , Proteómica/métodos , Pyrococcus furiosus/química , Proteínas Arqueales/genética , Clonación Molecular , Cristalografía/métodos , Difracción de Rayos X/métodosRESUMEN
Using a high degree of automation, the Southeast Collaboratory for Structural Genomics (SECSG) has developed high throughput pipelines for protein production, and crystallization using a two-tiered approach. Primary, or tier-1, protein production focuses on producing proteins for members of large Pfam families that lack a representative structure in the Protein Data Bank. Target genomes are Pyrococcus furiosus and Caenorhabditis elegans. Selected human proteins are also under study. Tier-2 protein production, or target rescue, focuses on those tier-1 proteins, which either fail to crystallize or give poorly diffracting crystals. This two tier approach is more efficient since it allows the primary protein production groups to focus on the production of new targets while the tier-2 efforts focus on providing additional sample for further studies and modified protein for structure determination. Both efforts feed the SECSG high throughput crystallization pipeline, which is capable of screening over 40 proteins per week. Details of the various pipelines in use by the SECSG for protein production and crystallization, as well as some examples of target rescue are described.
Asunto(s)
Caenorhabditis elegans/química , Cristalización/métodos , Bases de Datos de Proteínas , Proteínas/química , Proteínas/aislamiento & purificación , Proteómica/métodos , Pyrococcus furiosus/química , Animales , Humanos , Proteínas/metabolismo , Proteómica/instrumentación , Proteómica/organización & administraciónRESUMEN
The unique clostridial endo-beta-galactosidase (Endo-beta-Gal(GnGa)) capable of releasing the disaccharide GlcNAc alpha 1,4Gal from O-glycans expressed in the gastric gland mucous cell-type mucin has been crystallized. The crystal belongs to space group P6(3), with unit-cell parameters a = 160.4, c = 86.1 A. Under cryocooled conditions and using a synchrotron X-ray source, the crystals diffract to 1.82 A resolution. The asymmetric unit contains two or three molecules.