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1.
PLoS Pathog ; 16(9): e1008827, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886721

RESUMO

Global burden of cervical cancer, the most common cause of mortality caused by human papillomavirus (HPV), is expected to increase during the next decade, mainly because current alternatives for HPV vaccination and cervical cancer screening programs are costly to be established in low-and-middle income countries. Recently, we described the development of the broadly protective, thermostable vaccine antigen Trx-8mer-OVX313 based on the insertion of eight different minor capsid protein L2 neutralization epitopes into a thioredoxin scaffold from the hyperthermophilic archaeon Pyrococcus furiosus and conversion of the resulting antigen into a nanoparticle format (median radius ~9 nm) upon fusion with the heptamerizing OVX313 module. Here we evaluated whether the engineered thioredoxin scaffold, in addition to humoral immune responses, can induce CD8+ T-cell responses upon incorporation of MHC-I-restricted epitopes. By systematically examining the contribution of individual antigen modules, we demonstrated that B-cell and T-cell epitopes can be combined into a single antigen construct without compromising either immunogenicity. While CD8+ T-cell epitopes had no influence on B-cell responses, the L2 polytope (8mer) and OVX313-mediated heptamerization of the final antigen significantly increased CD8+ T-cell responses. In a proof-of-concept experiment, we found that vaccinated mice remained tumor-free even after two consecutive tumor challenges, while unvaccinated mice developed tumors. A cost-effective, broadly protective vaccine with both prophylactic and therapeutic properties represents a promising option to overcome the challenges associated with prevention and treatment of HPV-caused diseases.


Assuntos
Antígenos de Neoplasias , Antígenos Virais , Proteínas Arqueais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer , Imunidade Celular/efeitos dos fármacos , Nanopartículas , Papillomaviridae , Vacinas contra Papillomavirus , Pyrococcus furiosus/química , Tiorredoxinas , Neoplasias do Colo do Útero/imunologia , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/farmacologia , Antígenos Virais/química , Antígenos Virais/farmacologia , Proteínas Arqueais/química , Proteínas Arqueais/farmacologia , Linfócitos T CD8-Positivos/patologia , Vacinas Anticâncer/química , Vacinas Anticâncer/farmacologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/farmacologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Papillomaviridae/química , Papillomaviridae/imunologia , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/farmacologia , Tiorredoxinas/química , Tiorredoxinas/farmacologia , Neoplasias do Colo do Útero/virologia
2.
J Biotechnol ; 310: 68-79, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32014561

RESUMO

Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nano-biotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosusl-asparaginase, (NPfA, a protein chaperone lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. We tested the effect of NPfA on substrate proteins, ascorbate peroxidase (APX), IgG peroxidase antibodies (I-HAbs) and KOD DNA polymerase. Each protein not only displayed increased thermal stability but also increased activity in the presence of NPfA. This increase was either comparable or higher than those obtained by common osmolytes; glycine betaine, sorbitol and trehalose. Most dramatic activity enhancement was seen in the case of KOD polymerase (∼ 40 % increase). NPfA exerts its effect through transient binding to the substrate proteins as discerned through isothermal titration calorimetry, dynamic light scattering and size exclusion chromatography. Mechanistic insights obtained through simulations suggested a remodeled architecture and emergence of H-binding network between NPfA and substrate protein with an effective enhancement in the solvent accessibility at the active site pocket of the latter. Thus, the capability of NPfA to engage in specific manner with other proteins is demonstrated to reduce the concentration of substrate proteins/enzymes required per unit operation. The functional expansion obtained through our finding establishes NPfA as a novel class of ATP-independent molecular chaperone with immense future biotechnological applications.


Assuntos
Proteínas Arqueais/química , Asparaginase/química , Chaperonas Moleculares/química , Pyrococcus furiosus/química , Proteínas Arqueais/genética , Asparaginase/genética , Chaperonas Moleculares/genética , Plasmodium falciparum/química , Plasmodium falciparum/genética , Domínios Proteicos , Estabilidade Proteica , Pyrococcus furiosus/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Thermococcus/química , Thermococcus/genética
3.
Org Biomol Chem ; 18(10): 1881-1885, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32100807

RESUMO

A convenient two-step method is reported for the ligation of alkoxyamine- or hydrazine-bearing cargo to proline N-termini. Using this approach, bifunctional proline N-terminal bioconjugates are constructed and proline N-terminal proteins are immobilized.


Assuntos
Aminas/química , Hidrazinas/química , Prolina/química , Proteínas/síntese química , Hidrazonas/síntese química , Cetonas/síntese química , Oxirredução , Oximas/síntese química , Pyrococcus furiosus/química , Vírus do Mosaico do Tabaco/química
4.
Chem Commun (Camb) ; 55(88): 13219-13222, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31589231

RESUMO

We demonstrate that short single-stranded DNA generated by Pyrococcus furiosus Argonaute (PfAgo) can initiate a second round of cleavage. Based on this principle, we established a molecular diagnostic method, termed PfAgo-mediated Nucleic acid Detection (PAND). This method could detect DNA at attomolar sensitivities, distinguish single-nucleotide mutants and accomplish multiplexed detection.


Assuntos
DNA/análise , Pyrococcus furiosus/química , Humanos
5.
J Am Chem Soc ; 141(33): 13049-13056, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31356074

RESUMO

Multimeric enzyme complexes are ubiquitous in nature and catalyze a broad range of useful biological transformations. They are often characterized by a tight allosteric coupling between subunits, making them highly inefficient when isolated. A good example is Tryptophan synthase (TrpS), an allosteric heterodimeric enzyme in the form of an αßßα complex that catalyzes the biosynthesis of L-tryptophan. In this study, we decipher the allosteric regulation existing in TrpS from Pyrococcus furiosus (PfTrpS), and how the allosteric conformational ensemble is recovered in laboratory-evolved stand-alone ß-subunit variants. We find that recovering the conformational ensemble of a subdomain of TrpS affecting the relative stabilities of open, partially closed, and closed conformations is a prerequisite for enhancing the catalytic efficiency of the ß-subunit in the absence of its binding partner. The distal mutations resuscitate the allosterically driven conformational regulation and alter the populations and rates of exchange between these multiple conformational states, which are essential for the multistep reaction pathway of the enzyme. Interestingly, these distal mutations can be a priori predicted by careful analysis of the conformational ensemble of the TrpS enzyme through computational methods. Our study provides the enzyme design field with a rational approach for evolving allosteric enzymes toward improved stand-alone function for biosynthetic applications.


Assuntos
Pyrococcus furiosus/enzimologia , Triptofano Sintase/química , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Pyrococcus furiosus/química , Pyrococcus furiosus/metabolismo , Triptofano/metabolismo , Triptofano Sintase/metabolismo
6.
J Biol Chem ; 294(34): 12807-12814, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31289123

RESUMO

As a contributor to multidrug resistance, the family of multidrug and toxin extrusion (MATE) transporters couples the efflux of chemically dissimilar compounds to electrochemical ion gradients. Although divergent transport mechanisms have been proposed for these transporters, previous structural and functional analyses of members of the MATE subfamily DinF suggest that the N-terminal domain (NTD) supports substrate and ion binding. In this report, we investigated the relationship of ligand binding within the NTD to the drug resistance mechanism of the H+-dependent MATE from the hyperthermophilic archaeon Pyrococcus furiosus (PfMATE). To facilitate this study, we developed a cell growth assay in Escherichia coli to characterize the resistance conferred by PfMATE to toxic concentrations of the antimicrobial compound rhodamine 6G. Expression of WT PfMATE promoted cell growth in the presence of drug, but amino acid substitutions of conserved NTD residues compromised drug resistance. Steady-state binding analysis with purified PfMATE indicated that substrate affinity was unperturbed in these NTD variants. However, exploiting Trp fluorescence as an intrinsic reporter of conformational changes, we found that these variants impaired formation of a unique H+-stabilized structural intermediate. These results imply that disruption of H+ coupling is the origin of compromised toxin resistance in PfMATE variants. These findings support a model mechanism wherein the NTD mediates allosteric coupling to ion gradients through conformational changes to drive substrate transport in PfMATE. Furthermore, the results provide evidence for diverging transport mechanisms within a prokaryotic MATE subfamily.


Assuntos
Antineoplásicos/farmacologia , Proteínas Arqueais/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Prótons , Pyrococcus furiosus/química , Rodaminas/farmacologia , Proteínas Arqueais/química , Proliferação de Células/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/citologia , Pyrococcus furiosus/metabolismo
7.
Biochem J ; 476(6): 975-989, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30837306

RESUMO

Ferritins are a large family of intracellular proteins that protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. The recently characterized Rhodospirillum rubrum EncFtn displays an unusual structure when compared with classical ferritins, with an open decameric structure that is enzymatically active, but unable to store iron. This EncFtn must be associated with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether this unusual structure is conserved across the family. Here, we characterize EncFtn proteins from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the conserved annular pentamer of dimers topology. Key structural differences are apparent between the homologues, particularly in the centre of the ring and the secondary metal-binding site, which is not conserved across the homologues. Solution and native mass spectrometry analyses highlight that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The ferroxidase activity of EncFtn proteins was confirmed, and we show that while the quaternary structure around the ferroxidase centre is distinct from classical ferritins, the ferroxidase activity is still inhibited by Zn(II). Our results highlight the common structural organization and activity of EncFtn proteins, despite diverse host environments and contexts within encapsulins.


Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Ferritinas/química , Myxococcales/química , Pyrococcus furiosus/química , Rhodospirillum rubrum/química , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
8.
Methods Mol Biol ; 1873: 69-92, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30341604

RESUMO

Chaperonopathies are diseases in which abnormal chaperones play an etiopathogenic role. A chaperone is mutated or otherwise abnormal (e.g., modified by an aberrant posttranslational modification) in structure/function. To understand the pathogenic mechanisms of chaperonopathies, it is necessary to elucidate the impact of the pathogenic mutation or posttranslational modification on the chaperone molecule's properties and functions. This impact is usually subtle because if it were more than subtle the overall effect on the cell and organism would be catastrophic, lethal. This is because most chaperones are essential for life and, if damaged in structure/function too strongly, there would be death of the cell/organism, and no phenotype, i.e., there would be no patients with chaperonopathies. Consequently, diagnostic procedures and analysis of defects of the abnormal chaperones require a multipronged method for assessing the chaperone molecule from various angles. Here, we present such a method that includes assessing the intrinsic properties and the chaperoning functions of chaperone molecules.


Assuntos
Proteínas Arqueais/química , Varredura Diferencial de Calorimetria/métodos , Microscopia de Força Atômica/métodos , Chaperonas Moleculares/química , Mutação , Processamento de Proteína Pós-Traducional , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Temperatura Alta , Humanos , Malato Desidrogenase/química , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Penaeidae/química , Estabilidade Proteica , Pyrococcus furiosus/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
J Colloid Interface Sci ; 537: 20-27, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30415098

RESUMO

Palladium and silver nanoparticles (NPs) anchored at the outer surface of ferritin form stable suspension of non-coated particles that possess several catalytic and enzymomimetic activities. These activities are strongly affected by detergents that significantly influence the reaction efficiency and specificity. Reductive dehalogenation of various azo dye substrates shows strong differences in reactivity for each substrate-detergent pair. Reductive dehalogenation is negatively influenced by cationic detergents while catalytic depropargylation is severely impaired by polyethylene oxide containing detergents that is an important finding in respect to potential biorthogonal applications. Moreover, Suzuki-Miyaura reaction is promoted by polyethylene oxide containing detergents but some of them also facilitate dehalogenation. Enzymomimetic peroxidase activity of silver NPs can be detected only in presence of sodium dodecyl sulfate (SDS) while peroxidase activity of palladium NPs is enhanced by SDS and sodium deoxycholate.


Assuntos
Biomimética , Detergentes/química , Ferritinas/metabolismo , Nanopartículas Metálicas/química , Peroxidase/metabolismo , Pyrococcus furiosus/metabolismo , Prata/metabolismo , Catálise , Ferritinas/química , Paládio/química , Paládio/metabolismo , Tamanho da Partícula , Peroxidase/química , Pyrococcus furiosus/química , Prata/química , Propriedades de Superfície
10.
J Phys Chem B ; 123(2): 480-486, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30566356

RESUMO

In this simulation study, we investigate the influence of biomolecular confinement on dynamical processes in water. We compare water confined in a membrane protein nanopore at room temperature to pure liquid water at low temperatures with respect to structural relaxations, intermolecular vibrations, and the propagation of collective modes. We observe distinct potential energy landscapes experienced by water molecules in the two environments, which nevertheless result in comparable hydrogen bond lifetimes and sound propagation velocities. Hence, we show that a viscoelastic argument that links slow rearrangements of the water-hydrogen bond network to ice-like collective properties applies to both, the pure liquid and biologically confined water, irrespective of differences in the microscopic structure.


Assuntos
Proteínas Arqueais/química , Canais de Translocação SEC/química , Água/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Nanoporos , Pyrococcus furiosus/química , Temperatura
11.
Angew Chem Int Ed Engl ; 57(45): 14764-14768, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30215880

RESUMO

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and ß positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive ß-branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of ß-branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB7E6 . PfTrpB7E6 is the first biocatalyst to synthesize bulky ß-branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB7E6 was explored through X-ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active-site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB7E6 provides an operationally simple and environmentally benign platform for the preparation of ß-branched tryptophan building blocks.


Assuntos
Pyrococcus furiosus/enzimologia , Triptofano Sintase/metabolismo , Triptofano/análogos & derivados , Triptofano/metabolismo , Biocatálise , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Mutação , Engenharia de Proteínas , Pyrococcus furiosus/química , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Triptofano Sintase/química , Triptofano Sintase/genética
12.
Nucleic Acids Res ; 46(19): 10066-10081, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30102372

RESUMO

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.


Assuntos
Proteínas Arqueais/química , DNA/química , RNA Polimerase II/química , Fator de Transcrição TFIIB/química , Proteínas Arqueais/genética , DNA/genética , Domínios Proteicos , Pyrococcus furiosus/química , Pyrococcus furiosus/genética , RNA Polimerase II/genética , Fator de Transcrição TFIIB/genética , Transcrição Genética
13.
Nucleic Acids Res ; 46(17): 9027-9043, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30102394

RESUMO

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.


Assuntos
Proteínas Arqueais/química , Reparo do DNA , DNA de Cadeia Simples/química , Exonucleases/química , Pyrococcus furiosus/enzimologia , RNA Arqueal/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , Quebras de DNA de Cadeia Simples , Dano ao DNA , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Expressão Gênica , Haloferax volcanii/química , Haloferax volcanii/efeitos dos fármacos , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Cinética , Manganês/química , Manganês/metabolismo , Metanossulfonato de Metila/farmacologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pyrococcus furiosus/química , Pyrococcus furiosus/efeitos dos fármacos , Pyrococcus furiosus/genética , RNA Arqueal/genética , RNA Arqueal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
ACS Chem Biol ; 13(9): 2472-2483, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30060648

RESUMO

Single-molecule techniques allow unique insights into biological systems as they provide unrivaled access to structural dynamics and conformational heterogeneity. One major bottleneck for reliable single-molecule Förster resonance energy transfer (smFRET) analysis is the identification of suitable fluorophore labeling sites that neither impair the function of the biological system nor cause photophysical artifacts of the fluorophore. To address this issue, we identified the contribution of virtually all individual parameters that affect Förster resonance energy transfer between two fluorophores attached to a ribonucleoprotein complex consisting of the RNA-binding protein L7Ae and a cognate kink turn containing RNA. A non-natural amino acid was incorporated at various positions of the protein using an amber suppression system (pEVOL) to label the protein via copper(I)-catalyzed alkyne-azide cycloaddition. On the basis of simulations followed by functional, structural, and multiparameter fluorescence analysis of five different smFRET RNPs, new insights into the design of smFRET RNPs were obtained. From this, a correlation between the photophysical properties of fluorophores attached to the protein and the predictability of the corresponding smFRET construct was established. Additionally, we identify a straightforward experimental method for characterizing selected labeling sites. Overall, this protocol allows fast generation and assessment of functional RNPs for accurate single-molecule experiments.


Assuntos
Proteínas de Bactérias/química , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Pyrococcus furiosus/química , RNA/química , Ribonucleoproteínas/química , Fluorescência , Modelos Moleculares
15.
Chem Commun (Camb) ; 54(65): 8972-8975, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-29974085

RESUMO

Fast (>100 kHz) magic angle spinning solid-state NMR allows combining high-sensitive proton detection with the absence of an intrinsic molecular weight limit. Using this technique we observe for the first time narrow 1H RNA resonances and assign nucleotide spin systems with only 200 µg of uniformly 13C,15N-labelled RNA.


Assuntos
Proteínas Arqueais/química , Ressonância Magnética Nuclear Biomolecular/métodos , Prótons , Proteínas de Ligação a RNA/química , RNA/química , Isótopos de Carbono , Isótopos de Nitrogênio , Pyrococcus furiosus/química
16.
Proc Natl Acad Sci U S A ; 115(27): E6172-E6181, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29915058

RESUMO

Multidrug and toxic-compound extrusion (MATE) proteins comprise an important but largely uncharacterized family of secondary-active transporters. In both eukaryotes and prokaryotes, these transporters protect the cell by catalyzing the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are thus potential pharmacological targets against drug-resistant pathogenic bacteria and tumor cells. The activity of MATEs is powered by transmembrane electrochemical ion gradients, but their molecular mechanism and ion specificity are not understood, in part because high-quality structural information is limited. Here, we use computational methods to study PfMATE, from Pyrococcus furiosus, whose structure is the best resolved to date. Analysis of available crystallographic data and additional molecular dynamics simulations unequivocally reveal an occupied Na+-binding site in the N-lobe of this transporter, which had not been previously recognized. We find this site to be selective against K+ and broadly conserved among prokaryotic MATEs, including homologs known to be Na+-dependent such as NorM-VC, VmrA, and ClbM, for which the location of the Na+ site had been debated. We note, however, that the chemical makeup of the proposed Na+ site indicates it is weakly specific against H+, explaining why MATEs featuring this Na+-binding motif may be solely driven by H+ in laboratory conditions. We further posit that the concurrent coupling to H+ and Na+ gradients observed for some Na+-driven MATEs owes to a second H+-binding site, within the C-lobe. In summary, our study provides insights into the structural basis for the complex ion dependency of MATE transporters.


Assuntos
Proteínas Arqueais/química , Proteínas de Transporte/química , Pyrococcus furiosus/química , Motivos de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Relação Estrutura-Atividade
17.
Methods Enzymol ; 599: 409-425, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746248

RESUMO

For over 20 years, nuclear resonance vibrational spectroscopy (NRVS) has been used to study vibrational dynamics of iron-containing materials. With the only selection rule being iron motion, 57Fe NRVS has become an excellent tool to study iron-containing enzymes. Over the past decade, considerable progress has been made in the study of complex metalloenzymes using NRVS. Iron cofactors in heme-containing globins; [2Fe2S], [3Fe4S], [4Fe4S] proteins; the [NiFe] and [FeFe] hydrogenases; and nitrogenases have been explored in a fashion not possible through traditional vibrational spectroscopy. In this chapter, we discuss the basics of NRVS, a strategy to perform NRVS, and a discussion of the application of NRVS on rubredoxin and [FeFe] hydrogenase.


Assuntos
Proteínas com Ferro-Enxofre/química , Espectroscopia de Mossbauer/métodos , Proteínas Arqueais/química , Chlamydomonas reinhardtii/química , Hidrogenase/química , Modelos Moleculares , Oxirredução , Fótons , Proteínas de Plantas/química , Pyrococcus furiosus/química , Rubredoxinas/química , Software , Síncrotrons
18.
Protein Eng Des Sel ; 31(1): 29-36, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29301037

RESUMO

Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallographically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment.


Assuntos
Proteínas Arqueais/química , Ativadores de Enzimas/química , Chaperonas Moleculares/química , Mutação , Complexo de Endopeptidases do Proteassoma/química , Pyrococcus furiosus/química , Proteínas Arqueais/genética , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/genética , Domínios Proteicos , Pyrococcus furiosus/genética
19.
Biochemistry ; 57(6): 978-990, 2018 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-29303562

RESUMO

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.


Assuntos
Bactérias/química , Proteínas de Bactérias/química , Proteínas com Ferro-Enxofre/química , Azotobacter vinelandii/química , Domínio Catalítico , Transporte de Elétrons , Ferredoxinas/química , Modelos Moleculares , Oxirredução , Oxirredutases/química , Conformação Proteica , Pseudomonas putida/química , Pyrococcus furiosus/química , Rubredoxinas/química
20.
Protein Expr Purif ; 142: 25-31, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28963004

RESUMO

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni2+. Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli.


Assuntos
Escherichia coli/genética , Histidina/genética , Proteínas Ligantes de Maltose/genética , Oligopeptídeos/genética , Proteínas Recombinantes de Fusão/genética , Trissacarídeos/metabolismo , Cromatografia de Afinidade/métodos , Clonagem Molecular , Endopeptidases/química , Escherichia coli/metabolismo , Expressão Gênica , Histidina/isolamento & purificação , Histidina/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Pyrococcus furiosus/química , Pyrococcus furiosus/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Trissacarídeos/química , Trissacarídeos/isolamento & purificação
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