RESUMEN
HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin ß (Impß), but how Rev associates with Impß is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impß/Rev complex. We show that Impß binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impß cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impß-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impß with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impß and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impß cargos.
Asunto(s)
VIH-1 , beta Carioferinas , VIH-1/metabolismo , Modelos Estructurales , Simulación del Acoplamiento Molecular , ARN Viral/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen.
Asunto(s)
Mononucleótido de Flavina , Oxígeno Singlete , Mononucleótido de Flavina/química , Flavoproteínas/química , Oxígeno/química , Fosfatos , Riboflavina , Oxígeno Singlete/químicaRESUMEN
Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.
Asunto(s)
ADN/química , Histonas/química , Proteína 1 de Ensamblaje de Nucleosomas/química , Nucleosomas/ultraestructura , Animales , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Radical Hidroxilo/química , Modelos Moleculares , Proteína 1 de Ensamblaje de Nucleosomas/genética , Proteína 1 de Ensamblaje de Nucleosomas/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Concentración Osmolar , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
ECFP, the first usable cyan fluorescent protein (CFP), was obtained by adapting the tyrosine-based chromophore environment in green fluorescent protein to that of a tryptophan-based one. This first-generation CFP was superseded by the popular Cerulean, CyPet, and SCFP3A that were engineered by rational and random mutagenesis, yet the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the third-generation CFP mTurquoise and its successors without an obvious rationale. We show here that the evolution process had unexpectedly remodeled the chromophore environment in second-generation CFPs so they would accommodate a different isomer, whose formation is favored by acidic pH or light irradiation and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters and provide novel insights into the rational evolution of fluorescent proteins with a tryptophan-based chromophore.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Cristalografía por Rayos X , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Isomerismo , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Triptófano/química , Triptófano/genética , Triptófano/metabolismoRESUMEN
Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis.
Asunto(s)
Microscopía por Crioelectrón/métodos , Glutamato-Amoníaco Ligasa/química , Proteínas de Unión a Maltosa/química , Proteínas Recombinantes de Fusión/química , Multimerización de ProteínaRESUMEN
Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality.
Asunto(s)
Bacteriófagos/metabolismo , Histidina/metabolismo , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/metabolismo , Bacteriófagos/ultraestructura , Cromatografía en Gel , Clonación Molecular , Cristalización , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Espectroscopía de Resonancia Magnética , Solubilidad , Proteínas Virales/aislamiento & purificaciónRESUMEN
Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.
Asunto(s)
Neoplasias Encefálicas/diagnóstico , Colorantes Fluorescentes/metabolismo , Proteínas Luminiscentes/metabolismo , Neuroimagen/métodos , Neuronas/metabolismo , Animales , Biliverdina/metabolismo , Neoplasias Encefálicas/metabolismo , Cristalografía por Rayos X , Drosophila , Células HEK293 , Hemo Oxigenasa (Desciclizante)/metabolismo , Humanos , Rayos Infrarrojos , Larva , Ratones , Microscopía Confocal , Fitocromo , RatasRESUMEN
There is increasing interest in applying NMR spectroscopy to the study of large protein assemblies. Development of methyl-specific labeling protocols combined with improved NMR spectroscopy enable nowadays studies of proteins complexes up to 1 MDa. For such large complexes, the major interest lies in obtaining structural, dynamic and interaction information in solution, which requires sequence-specific resonance assignment of NMR signals. While such analysis is quite standard for small proteins, it remains one of the major bottlenecks when the size of the protein increases. Here, we describe implementation and latest improvements of SeSAM, a fast and user-friendly approach for assignment of methyl resonances in large proteins using mutagenesis. We have improved culture medium to boost the production of methyl-specifically labeled proteins, allowing us to perform small-scale parallel production and purification of a library of (13)CH3-specifically labeled mutants. This optimized protocol is illustrated by assignment of Alanine, Isoleucine, and Valine methyl groups of the homododecameric aminopeptidase PhTET2. We estimated that this improved method allows assignment of ca. 100 methyl cross-peaks in 2 weeks, including 4 days of NMR time and less than 2 k of isotopic materials.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Aminoácidos/química , Biblioteca de Genes , Marcaje Isotópico , Peso Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/genéticaRESUMEN
The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , División Celular , Pared Celular/metabolismo , Proteínas de la Membrana/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Hidrólisis , Cinética , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/genéticaRESUMEN
Cryoprotection of a protein crystal by addition of small-molecule compounds may sometimes affect the structure of its active site. The spectroscopic and structural effects of the two cryoprotectants glycerol and ethylene glycol on the cyan fluorescent protein Cerulean were investigated. While glycerol had almost no noticeable effect, ethylene glycol was shown to induce a systematic red shift of the UV-vis absorption and fluorescence emission spectra. Additionally, ethylene glycol molecules were shown to enter the core of the protein, with one of them binding in close vicinity to the chromophore, which provides a sound explanation for the observed spectroscopic changes. These results highlight the need to systematically record spectroscopic data on crystals of light-absorbing proteins and reinforce the notion that fluorescent proteins must not been seen as rigid structures.
Asunto(s)
Crioprotectores/química , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Hidrozoos/química , Animales , Crioprotectores/metabolismo , Cristalografía por Rayos X , Glicol de Etileno/química , Glicol de Etileno/metabolismo , Colorantes Fluorescentes/metabolismo , Glicerol/química , Glicerol/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hidrozoos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
The green fluorescent protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan fluorescent protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Hidrozoos/química , Mutación , Animales , Proteínas Fluorescentes Verdes/genética , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
To make progress in understanding peptidoglycan metabolism, we will reconstitute in vitro the assembly process and the molecular machineries that carry out this formidable task. We review here the reports of isolation of complexes comprising penicillin-binding proteins (PBPs), the enzymes that synthesize the peptidoglycan from its lipid-linked precursor.
Asunto(s)
Pared Celular/metabolismo , Escherichia coli/metabolismo , Peptidoglicano/biosíntesis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Pared Celular/química , Pared Celular/genética , Cromatografía de Afinidad , Escherichia coli/genética , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Inmunoprecipitación , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Coloración y EtiquetadoRESUMEN
Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh ß-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Proteínas Bacterianas/química , Línea Celular Tumoral , Cristalografía por Rayos X/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/ultraestructura , Células HeLa , Humanos , Enlace de Hidrógeno , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.
Asunto(s)
Mutagénesis Sitio-Dirigida/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Enhanced Green Fluorescent Protein (EGFP) is a variant of wild-type Green Fluorescent Protein from the jellyfish Aequorea victoria, whose mutations S65T and F64L increase brightness and folding efficiency. EGFP is extensively used in cell biology and biochemistry as a colocalization or expression reporter. Surprisingly, the structure of this very popular protein has not been determined yet. We report here its crystallographic structure at 1.5Å resolution which shows significant differences in the vicinity of residue 64 and of the chromophore. In particular, two conformations are observed for the key residue glutamic acid 222, in apparent contradiction with the single fluorescence lifetime of the protein. We then show that X-ray induced decarboxylation of Glu222 during diffraction data collection results in the disruption of a hydrogen-bond network near the chromophore. Using single-crystal microspectrophotometry, we demonstrate that this correlates with a significant loss of the fluorescence properties. We thus propose a mechanism of bleaching of the protein at low temperature. Taken together, these two sets of results highlight the stabilizing role of Glu222 to the chromophore cavity of EGFP.
Asunto(s)
Ácido Glutámico/química , Proteínas Fluorescentes Verdes/química , Animales , Cristalografía por Rayos X , Fluorescencia , Ácido Glutámico/efectos de la radiación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/efectos de la radiación , Hydra , Mutación , Estabilidad Proteica , Estructura Terciaria de ProteínaRESUMEN
In this report we describe a rapid, simple, and efficient method for large-scale purification of linear plasmid DNA to answer demand from high-throughput gene cloning. The process is based on the separation of the linear vector from small DNA fragments by anion exchange chromatography. Gene cloning experiments by restriction/ligation or the In-Fusion technique confirmed the high quality of the linearized vector as 100% of the genes were successfully cloned.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Clonación Molecular , Fragmentación del ADN , Proteínas de la Membrana/genética , Plásmidos/aislamiento & purificación , Escherichia coli/genética , Vectores Genéticos , Proteínas de la Membrana/metabolismo , Resinas SintéticasRESUMEN
BACKGROUND: Streptococcus pneumoniae is a widely distributed commensal Gram-positive bacteria of the upper respiratory tract. Pneumococcal colonization can progress to invasive disease, and thus become lethal, reason why antibiotics and vaccines are designed to limit the dramatic effects of the bacteria in such cases. As a consequence, pneumococcus has developed efficient antibiotic resistance, and the use of vaccines covering a limited number of serotypes such as Pneumovax and Prevnar results in the expansion of non-covered serotypes. Pneumococcal surface proteins represent challenging candidates for the development of new therapeutic targets against the bacteria. Despite the number of described virulence factors, we believe that the majority of them remain to be characterized. This is the reason why pneumococcus invasion processes are still largely unknown. RESULTS: Availability of genome sequences facilitated the identification of pneumococcal surface proteins bearing characteristic motifs such as choline-binding proteins (Cbp) and peptidoglycan binding (LPXTG) proteins. We designed a medium throughput approach to systematically test for interactions between these pneumococcal surface proteins and host proteins (extracellular matrix proteins, circulating proteins or immunity related proteins). We cloned, expressed and purified 28 pneumococcal surface proteins. Interactions were tested in a solid phase assay, which led to the identification of 23 protein-protein interactions among which 20 are new. CONCLUSIONS: We conclude that whether peptidoglycan binding proteins do not appear to be major adhesins, most of the choline-binding proteins interact with host proteins (elastin and C reactive proteins are the major Cbp partners). These newly identified interactions open the way to a better understanding of host-pneumococcal interactions.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Infecciones Neumocócicas/metabolismo , Streptococcus pneumoniae/metabolismo , Adhesinas Bacterianas/genética , Proteínas Bacterianas/genética , Humanos , Proteínas de la Membrana/genética , Infecciones Neumocócicas/microbiología , Unión Proteica , Streptococcus pneumoniae/genéticaRESUMEN
Enhanced cyan fluorescent protein (ECFP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.
Asunto(s)
Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Simulación por Computador , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Triptófano/químicaRESUMEN
A major challenge in structural biology remains the identification of protein constructs amenable to structural characterization. Here, we present a simple method for parallel expression, labeling, and purification of protein constructs (up to 80 kDa) combined with rapid evaluation by NMR spectroscopy. Our approach, which is equally applicable for manual or automated implementation, offers an efficient way to identify and optimize protein constructs for NMR or X-ray crystallographic investigations.