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1.
Protein Expr Purif ; 188: 105954, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34416360

RESUMO

Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling - perdeuteration - requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.


Assuntos
Deutério/química , Escherichia coli/genética , Difração de Nêutrons/métodos , Nêutrons , Porinas/química , Difração de Raios X/métodos , Clorófitas/química , Clorófitas/crescimento & desenvolvimento , Clonagem Molecular , Misturas Complexas/química , Cristalografia por Raios X/métodos , Meios de Cultura/química , Meios de Cultura/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Porinas/genética , Porinas/isolamento & purificação , Porinas/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
2.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 11): 536-543, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33135672

RESUMO

Serratia marcescens is an opportunistic pathogen that commonly causes hospital-acquired infections and can utilize chitin-enriched nutrients as an alternative energy source. This study reports the identification of a chitoporin (ChiP), termed SmChiP, from the outer membrane of S. marcescens. Sequence alignment with genetically characterized ChiPs suggests that SmChiP is more closely related to the monomeric EcChiP from Escherichia coli than to the trimeric VhChiP from Vibrio campbellii. A single crystal of SmChiP grown under the condition 22%(w/v) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0 diffracted X-ray synchrotron radiation to 1.85 Šresolution. SmChiP co-crystallized with chitohexaose under the condition 19%(w/v) PEG 1500, 2 M ammonium phosphate monobasic, 0.1 M HEPES pH 7.0 diffracted X-rays to 2.70 Šresolution. Preliminary crystallographic analysis shows that both SmChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groups P42212 and P41212, respectively. The SmChiP crystal has unit-cell parameters a = 82.97, b = 82.97, c = 189.53 Å, α = ß = γ = 90°, while the crystal of SmChiP in complex with chitohexaose has unit-cell parameters a = 73.24, b = 73.24, c = 213.46 Å, α = ß = γ = 90°. Initial assessment of the complex structure clearly revealed electron density for the sugar ligand. Structure determination of SmChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization by S. marcescens.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Porinas/química , Serratia marcescens/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Cristalização , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Humanos , Oligossacarídeos/química , Porinas/genética , Porinas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Serratia marcescens/genética , Espectrometria de Massas por Ionização por Electrospray
3.
Molecules ; 25(14)2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32650591

RESUMO

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.


Assuntos
Organismos Aquáticos/química , Proteínas de Bactérias , Marinomonas/química , Porinas , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Porinas/química , Porinas/isolamento & purificação
4.
Biologicals ; 62: 22-26, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31668855

RESUMO

Salmonella is found to be a major causes of food borne diseases globally. Poultry products contaminated with this pathogen is one of the major sources of infections in humans. Outer membrane protein C (OmpC) of Salmonella Typhimurium is a promising DNA vaccine candidate to mitigate Salmonella infection in poultry. However, the large-scale production of bioactive recombinant OmpC (rOmpC) protein is hindered due to the formation of inclusion bodies in Escherichia coli. The objective of this work was to attain high level expression of rOmpC protein, purify and evaluate its functional properties. The ompC gene was optimized and fused with small ubiquitin-related modifier (SUMO) gene for high level expression as soluble protein. The fusion protein with ~58 kDa molecular weight was observed on SDS-PAGE gel. The expression levels of rOmpC fusion protein reached maximum of 38% of total soluble protein (TSP) after 8 h of 0.2% rhamnose induction. Protein purification was carried out using nickel nitrilotriacetic acid (Ni-NTA) purification column. Western blot were performed to analyse expression and immunoreactivity of rOmpC fusion protein. The results indicate that SUMO fusion system is ideal for large scale production of functional rOmpC fusion protein expression in E. coli.


Assuntos
Proteínas de Bactérias , Escherichia coli , Imunoglobulinas/imunologia , Porinas , Proteínas Recombinantes de Fusão , Proteína SUMO-1 , Salmonella typhimurium/genética , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Galinhas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Porinas/biossíntese , Porinas/genética , Porinas/imunologia , Porinas/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína SUMO-1/biossíntese , Proteína SUMO-1/genética , Proteína SUMO-1/imunologia , Proteína SUMO-1/isolamento & purificação , Salmonella typhimurium/metabolismo
5.
J Appl Microbiol ; 127(6): 1646-1655, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31529560

RESUMO

AIMS: The outer membrane porin protein (OMPP) of Bordetella bronchiseptica is an important adhesion factor and protective immunogen. The aim of this study was to verify the immunogenicity of recombinant OMPP and its protective efficacy against a lethal challenge with B. bronchiseptica in rabbits. METHODS AND RESULTS: Soluble rOMPP was successfully expressed in Escherichia coli, and the purified recombinant protein was mixed with the ISA 201 VG adjuvant to prepare a subunit vaccine for B. bronchiseptica. Rabbits were immunized with the rOMPP subunit vaccine and then infected with the virulent B. bronchiseptica strain QDBb01. Rabbits immunized with the subunit vaccine were completely protected compared to the control group, and the protective effect was obviously better than that of the inactivated whole-cell vaccine. Moreover, analysis of the immunization duration showed that the rOMPP subunit vaccine provided immune protection for at least 4 months after the second immunization. CONCLUSIONS: The rOMPP subunit vaccine completely protected rabbits from a subsequent B. bronchiseptica challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will provide key information for the development of a safe and effective recombinant subunit vaccine against B. bronchiseptica in rabbits.


Assuntos
Vacinas Bacterianas/imunologia , Infecções por Bordetella/prevenção & controle , Bordetella bronchiseptica/imunologia , Porinas/imunologia , Adjuvantes Imunológicos , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/isolamento & purificação , Infecções por Bordetella/imunologia , Bordetella bronchiseptica/patogenicidade , Imunização , Porinas/genética , Porinas/isolamento & purificação , Porinas/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vacinas de Subunidades Antigênicas
6.
Mar Drugs ; 16(4)2018 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-29621159

RESUMO

A diverse range of predatory marine gastropods produce toxins, yet most of these molecules remain uncharacterized. Conus species have received the most attention from researchers, leading to several conopeptides reaching clinical trials. This review aims to summarize what is known about bioactive compounds isolated from species of neglected marine gastropods, especially in the Turridae, Terebridae, Babyloniidae, Muricidae, Buccinidae, Colubrariidae, Nassariidae, Cassidae, and Ranellidae families. Multiple species have been reported to contain bioactive compounds with potential toxic activity, but most of these compounds have not been characterized or even clearly identified. The bioactive properties and potential applications of echotoxins and related porins from the Ranellidae family are discussed in more detail. Finally, the review concludes with a call for research on understudied species.


Assuntos
Organismos Aquáticos/química , Produtos Biológicos/química , Conotoxinas/química , Caramujo Conus/química , Porinas/química , Animais , Organismos Aquáticos/classificação , Organismos Aquáticos/fisiologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Pesquisa Biomédica/tendências , Biotecnologia/métodos , Biotecnologia/tendências , Classificação , Conotoxinas/isolamento & purificação , Conotoxinas/farmacologia , Caramujo Conus/classificação , Caramujo Conus/fisiologia , Conformação Molecular , Porinas/isolamento & purificação , Porinas/farmacologia , Comportamento Predatório
7.
Acta Crystallogr D Struct Biol ; 74(Pt 12): 1192-1199, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605133

RESUMO

Detergent micelles can solubilize membrane proteins, but there is always a need for a pool of free detergent at the critical micellar concentration to maintain the micelle-monomer equilibrium. Amphipol polymeric surfactants (APols) have been developed to replace conventional detergents in membrane-protein studies, but the role of free amphipol is unclear. It has previously been shown that the removal of free APol causes monodisperse outer membrane protein F (OmpF) to form long filaments. However, any remaining APol could not be resolved using electron microscopy. Here, small-angle neutron scattering with isotope contrast matching was used to separately determine the distributions of membrane protein and amphipol in a mixed sample. The data showed that after existing free amphipol had been removed from monodisperse complexes, a new equilibrium was established between protein-amphipol filaments and a pool of newly liberated free amphipol. The filaments consisted of OmpF proteins surrounded by a belt of Apol, whilst free oblate spheroid micelles of Apol were also present. No indications of long-range order were observed, suggesting a lack of defined structure in the filaments.


Assuntos
Escherichia coli/química , Micelas , Difração de Nêutrons/métodos , Porinas/química , Espalhamento a Baixo Ângulo , Tensoativos/análise , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Polímeros/análise , Porinas/isolamento & purificação , Conformação Proteica
8.
Biochemistry (Mosc) ; 82(11): 1304-1313, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29223157

RESUMO

Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8 M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8 M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8 M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8 M urea (pH 8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH 5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.


Assuntos
Porinas/química , Yersinia pseudotuberculosis/química , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias , Escherichia coli/genética , Escherichia coli/metabolismo , Corpos de Inclusão , Porinas/biossíntese , Porinas/isolamento & purificação , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacos , Proteínas Recombinantes , Soluções/química , Soluções/farmacologia , Água
9.
BMC Infect Dis ; 17(1): 485, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28693438

RESUMO

BACKGROUND: Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay). METHOD: Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23). RESULT: The rOmp2a was found to be immunoreactive by iELISA and western blotting. The test showed a sensitivity of 93.75% and specificity of 95.83% when tested against 185 serum samples. For determination of statistical significance between experimental groups and control groups, Student's t test was performed on the data. CONCLUSION: Omp2a emerges as a potential antigen candidate for serodiagnosis of human brucellosis.


Assuntos
Proteínas de Bactérias/imunologia , Brucelose/diagnóstico , Porinas/imunologia , Testes Sorológicos/métodos , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Brucella/imunologia , Brucella/patogenicidade , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Índia , Camundongos , Porinas/genética , Porinas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e Especificidade
10.
Gac Med Mex ; 152(Suppl 2): 5-13, 2016 Oct.
Artigo em Espanhol | MEDLINE | ID: mdl-27792711

RESUMO

In the present work, we report, for the first time, on the purification of the Salmonella Typhimurium OmpD porin. We assessed the integrity and purity of the protein and evaluated the immunogenicity of the protein and its ability to induce antibody without exogenous adjuvant. We observed that 10 µg OmpD induced high antibody levels of IgM and IgG, which were maintained for more than 260 days after immunization. Immunization with OmpD induced multiple IgG antibody isotypes including IgG1, IgG2a, IgG2b, and IgG3 subclasses. Furthermore, these antibodies were able to recognize and bind to the bacterial surface. Our results demonstrate the high immunogenicity of S. Typhimurium OmpD porin, which induces long-lasting antibodies which may be and important target of the immune response against Salmonella infection. In conclusion, we propose the OmpD porin could be used within novel subunit vaccine formulations that do not need additional adjuvant and that confer long lasting humoral immunity against Salmonella infections.


Assuntos
Anticorpos Antibacterianos/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Porinas/imunologia , Porinas/isolamento & purificação , Salmonella typhimurium/imunologia , Animais , Afinidade de Anticorpos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas contra Salmonella/imunologia
11.
FEMS Microbiol Lett ; 363(7)2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26912121

RESUMO

The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.


Assuntos
Mutação , Mycobacterium smegmatis/genética , Porinas/genética , Porinas/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Permeabilidade da Membrana Celular , Deleção de Genes , Genoma Bacteriano , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Nanoporos , Porinas/química , Porinas/fisiologia , Multimerização Proteica , Análise de Sequência de DNA
12.
FEBS Lett ; 590(2): 224-31, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26823169

RESUMO

This study was undertaken to characterize functions of the outer membrane protein OmpW, which potentially contributes to the development of colistin- and imipenem-resistance in Acinetobacter baumannii. Reconstitution of OmpW in artificial lipid bilayers showed that it forms small channels (23 pS in 1 m KCl) and markedly interacts with iron and colistin, but not with imipenem. In vivo, (55) Fe uptake assays comparing the behaviours of ΔompW mutant and wild-type strains confirmed a role for OmpW in A. baumannii iron homeostasis. However, the loss of OmpW expression did not have an impact on A. baumannii susceptibilities to colistin or imipenem.


Assuntos
Acinetobacter baumannii/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Colistina/metabolismo , Ferro/metabolismo , Porinas/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Dados de Sequência Molecular , Porinas/química , Porinas/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
13.
Biophys J ; 109(7): 1429-38, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26445443

RESUMO

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Porinas/química , Porinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Potenciais da Membrana/fisiologia , Membranas Artificiais , Simulação de Dinâmica Molecular , Mutação , Polifosfatos/química , Polifosfatos/metabolismo , Porinas/genética , Porinas/isolamento & purificação , Cloreto de Potássio/metabolismo , Conformação Proteica , Pseudomonas aeruginosa , Especificidade por Substrato
14.
Methods Enzymol ; 557: 149-66, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25950964

RESUMO

OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric ß-barrel made of 14 antiparallel ß-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring ß-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Proteínas de Escherichia coli/química , Escherichia coli/química , Porinas/química , Redobramento de Proteína , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Cromatografia em Gel , Cromatografia por Troca Iônica , Dicroísmo Circular , Cristalização/métodos , Eletroforese , Escherichia coli/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Modelos Moleculares , Porinas/isolamento & purificação , Porinas/metabolismo , Porinas/ultraestrutura , Estrutura Secundária de Proteína
15.
Appl Biochem Biotechnol ; 175(6): 2907-15, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25575589

RESUMO

Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.


Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Cromatografia em Gel/métodos , Haemophilus influenzae/química , Neisseria meningitidis/química , Porinas/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Fusobacterium nucleatum/química , Fusobacterium nucleatum/metabolismo , Haemophilus influenzae/metabolismo , Neisseria meningitidis/metabolismo , Porinas/química , Porinas/metabolismo , Estabilidade Proteica
16.
PLoS One ; 9(12): e114864, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25517996

RESUMO

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-ß-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.


Assuntos
Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Fracionamento Químico/métodos , Detergentes/química , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Bacillus subtilis , Proteínas de Bactérias/química , Cristalização , Escherichia coli/genética , Maltose/química , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/isolamento & purificação , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Porinas/isolamento & purificação , Estrutura Quaternária de Proteína
17.
Metallomics ; 6(7): 1254-68, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24870224

RESUMO

Marine cyanobacteria make a significant contribution to primary production whilst occupying some of the most nutrient poor regions of the world's oceans. The low bioavailability of trace metals can limit the growth of phytoplankton in ocean waters, but only scarce data are available on the requirements of marine microbes for zinc. Recent genome mining studies suggest that marine cyanobacteria have both uptake systems for zinc and proteins that utilize zinc as a cofactor. In this study, the oligotrophic strain Synechococcus sp. WH8102 was grown at different zinc concentrations. Using metalloproteomics approaches, we demonstrate that even though this organism's growth was not affected by extremely low zinc levels, cells accumulated significant quantities of zinc, which was shown to be protein-associated by 2D liquid chromatography and ICP-MS. This indicates that the mechanisms for zinc uptake in Synechococcus sp. WH8102 are extremely efficient. Significantly, expression of SYNW2224, a putative porin, was up-regulated during growth in zinc-depleted conditions. Furthermore, along with 30 other proteins, SYNW2224 was captured by immobilised zinc affinity chromatography, indicating the presence of surface-exposed site(s) with metal-binding capacity. It is proposed that this porin plays a role in high-affinity zinc uptake in this and other cyanobacteria.


Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte de Cátions/isolamento & purificação , Porinas/isolamento & purificação , Zinco/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte de Cátions/metabolismo , Cromatografia de Afinidade , Modelos Moleculares , Synechococcus
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 29(12): 1322-6, 2013 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-24321079

RESUMO

OBJECTIVE: To express and purify the recombinant major outer membrane protein (MOMP) of Legionella pneumophila (Lp) as diagnostic antigen, and explore its practical value in the serological diagnosis of Lp infection. METHODS: The recombinant plasmid pET-momp was transformed into the E.coli BL21 competent cells. The recombinant MOMP was induced to express, and then analyzed by SDS-PAGE electrophoresis, purified by affinity chromatography. We screened and obtained 58 positive blood serum and 32 negative blood serum using the DRG (Germany, IgG/IgM/IgA) Lp kit. The blood serum samples were detected for IgG, IgM, IgA antibody levels by indirect ELISA that we had established with the purified MOMP as the coating antigen, as well as by R&D (USA, IgG/IgM/IgA) Lp kit. Then using the receiver operating characteristic (ROC) curve, we compared these two methods in the sensitivity, specificity and consistency of the test results, for evaluating the application value of the indirect ELISA of recombinant MOMP. RESULTS: The approximately 45 000 recombinant MOMP was successfully expressed and purified. Compared with the indirect ELISA we established with the R&D Lp kit for detecting Lp antibody IgG, IgM and IgA in blood serum, the sensitivity of the indirect ELISA of recombinant MOMP to IgG was 90.9%, the specificity was 91.7%, the Kappa value was 0.784 (P < 0.05), and the area under the ROC curve was 0.913; the sensitivity to IgM was 91.4% and the specificity was 90.6%, the Kappa value was 0.809 (P < 0.05), and the area under the ROC curve was 0.910; the sensitivity to IgA was 92.1% and the specificity was 88.9%, the Kappa value was 0.793(P < 0.05), and the area under the ROC curve was 0.905. CONCLUSION: The recombinant MOMP was successfully induced to express and purified. The indirect ELISA we established with the recombinant MOMP protein as a diagnostic antigen showed good specificity, sensitivity and consistency, which laid a foundation for the development of serological diagnosis kit of Legionnaires' disease.


Assuntos
Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Legionella pneumophila , Porinas/imunologia , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Humanos , Isotipos de Imunoglobulinas/sangue , Porinas/genética , Porinas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
19.
PLoS One ; 8(11): e78272, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223145

RESUMO

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than 'normal' Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80-90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.


Assuntos
Proteínas de Bactérias/química , Borrelia burgdorferi/química , Membrana Celular/química , Bicamadas Lipídicas/química , Porinas/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Condutividade Elétrica , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Bicamadas Lipídicas/metabolismo , Maltose/química , Potenciais da Membrana/fisiologia , Oligossacarídeos/química , Polietilenoglicóis/química , Porinas/isolamento & purificação , Porinas/metabolismo , Cloreto de Potássio/química , Multimerização Proteica
20.
Biochemistry (Mosc) ; 78(5): 496-504, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23848152

RESUMO

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the "warm" variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the "cold" variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 ß-strands connected by short "periplasmic" and longer "extracellular" loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the "extracellular" loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Porinas/genética , Porinas/metabolismo , Yersinia enterocolitica/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Porinas/química , Porinas/isolamento & purificação , Estrutura Secundária de Proteína , Alinhamento de Sequência , Yersinia enterocolitica/química , Yersinia enterocolitica/genética
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