Base de dados : MEDLINE
Pesquisa : D12.776.463 [Categoria DeCS]
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  1 / 1915 MEDLINE  
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[PMID]:29367659
[Au] Autor:Martin LJ; Akhavan B; Bilek MMM
[Ad] Endereço:School of Physics, University of Sydney, Sydney, NSW, 2006, Australia.
[Ti] Título:Electric fields control the orientation of peptides irreversibly immobilized on radical-functionalized surfaces.
[So] Source:Nat Commun;9(1):357, 2018 01 24.
[Is] ISSN:2041-1723
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Surface functionalization of an implantable device with bioactive molecules can overcome adverse biological responses by promoting specific local tissue integration. Bioactive peptides have advantages over larger protein molecules due to their robustness and sterilizability. Their relatively small size presents opportunities to control the peptide orientation on approach to a surface to achieve favourable presentation of bioactive motifs. Here we demonstrate control of the orientation of surface-bound peptides by tuning electric fields at the surface during immobilization. Guided by computational simulations, a peptide with a linear conformation in solution is designed. Electric fields are used to control the peptide approach towards a radical-functionalized surface. Spontaneous, irreversible immobilization is achieved when the peptide makes contact with the surface. Our findings show that control of both peptide orientation and surface concentration is achieved simply by varying the solution pH or by applying an electric field as delivered by a small battery.
[Mh] Termos MeSH primário: Materiais Biocompatíveis/química
Proteínas Imobilizadas/química
Peptídeos/química
Titânio/química
[Mh] Termos MeSH secundário: Adsorção
Sequência de Aminoácidos
Eletricidade
Eletrodos Implantados
Seres Humanos
Concentração de Íons de Hidrogênio
Neuroestimuladores Implantáveis
Propriedades de Superfície
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Biocompatible Materials); 0 (Immobilized Proteins); 0 (Peptides); D1JT611TNE (Titanium)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180126
[St] Status:MEDLINE
[do] DOI:10.1038/s41467-017-02545-6


  2 / 1915 MEDLINE  
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[PMID]:28470616
[Au] Autor:Gräslund S; Savitsky P; Müller-Knapp S
[Ad] Endereço:Structural Genomics Consortium, Department of Biochemistry and Biophysics, Karolinska Institutet, Tomtebodavägen 23a, Gamma:6, 171 65, Solna, Sweden. susanne.graslund@ki.se.
[Ti] Título:In Vivo Biotinylation of Antigens in E. coli.
[So] Source:Methods Mol Biol;1586:337-344, 2017.
[Is] ISSN:1940-6029
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Site-specific biotinylation of proteins is often the method of choice to enable efficient immobilization of a protein on a surface without interfering with protein folding. The tight interaction of biotin and streptavidin is frequently used to immobilize an antigen during phage display selections of binders. Here we describe a method of in vivo biotinylation of proteins during expression in E. coli, by tagging the protein with the short biotin acceptor peptide sequence, Avi tag, and co-expression of the E. coli biotin ligase (BirA) resulting in precise biotinylation of a specific lysine residue in the tag.
[Mh] Termos MeSH primário: Antígenos/química
Antígenos/genética
Escherichia coli/genética
Proteínas Imobilizadas/química
Proteínas Imobilizadas/genética
[Mh] Termos MeSH secundário: Animais
Biotina/química
Biotinilação
Carbono-Nitrogênio Ligases/química
Carbono-Nitrogênio Ligases/genética
Clonagem Molecular/métodos
Escherichia coli/química
Proteínas de Escherichia coli/química
Proteínas de Escherichia coli/genética
Expressão Gênica
Vetores Genéticos/genética
Seres Humanos
Proteínas Repressoras/química
Proteínas Repressoras/genética
Estreptavidina/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antigens); 0 (Escherichia coli Proteins); 0 (Immobilized Proteins); 0 (Repressor Proteins); 6SO6U10H04 (Biotin); 9013-20-1 (Streptavidin); EC 6.3.- (Carbon-Nitrogen Ligases); EC 6.3.4.15 (birA protein, E coli)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180220
[Lr] Data última revisão:
180220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170505
[St] Status:MEDLINE
[do] DOI:10.1007/978-1-4939-6887-9_22


  3 / 1915 MEDLINE  
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[PMID]:28870733
[Au] Autor:Stenger B; Gerber A; Bernhardt R; Hannemann F
[Ad] Endereço:Institute of Biochemistry, Saarland University, Saarbrücken, Germany.
[Ti] Título:Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts.
[So] Source:Biochim Biophys Acta;1866(1):52-59, 2018 01.
[Is] ISSN:0006-3002
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
[Mh] Termos MeSH primário: Ácido 3-Hidroxibutírico/química
Bacillus megaterium/genética
Biotecnologia/métodos
Enzima de Clivagem da Cadeia Lateral do Colesterol/química
Proteínas Imobilizadas/biossíntese
Proteínas Mitocondriais/biossíntese
[Mh] Termos MeSH secundário: Ácido 3-Hidroxibutírico/biossíntese
Animais
Bacillus megaterium/enzimologia
Biocatálise
Bovinos
Colesterol/química
Colesterol/metabolismo
Enzima de Clivagem da Cadeia Lateral do Colesterol/genética
Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo
Grânulos Citoplasmáticos/química
Liofilização
Expressão Gênica
Proteínas Imobilizadas/química
Proteínas Imobilizadas/genética
Proteínas Mitocondriais/química
Proteínas Mitocondriais/genética
Pregnenolona/biossíntese
Pregnenolona/química
Refrigeração
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Immobilized Proteins); 0 (Mitochondrial Proteins); 73R90F7MQ8 (Pregnenolone); 97C5T2UQ7J (Cholesterol); EC 1.14.15.6 (Cholesterol Side-Chain Cleavage Enzyme); TZP1275679 (3-Hydroxybutyric Acid)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180208
[Lr] Data última revisão:
180208
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170906
[St] Status:MEDLINE


  4 / 1915 MEDLINE  
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[PMID]:28625736
[Au] Autor:Ducharme J; Auclair K
[Ad] Endereço:Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada.
[Ti] Título:Use of bioconjugation with cytochrome P450 enzymes.
[So] Source:Biochim Biophys Acta;1866(1):32-51, 2018 01.
[Is] ISSN:0006-3002
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Bioconjugation, defined as chemical modification of biomolecules, is widely employed in biological and biophysical studies. It can expand functional diversity and enable applications ranging from biocatalysis, biosensing and even therapy. This review summarizes how chemical modifications of cytochrome P450 enzymes (P450s or CYPs) have contributed to improving our understanding of these enzymes. Genetic modifications of P450s have also proven very useful but are not covered in this review. Bioconjugation has served to gain structural information and investigate the mechanism of P450s via photoaffinity labeling, mechanism-based inhibition (MBI) and fluorescence studies. P450 surface acetylation and protein cross-linking have contributed to the investigation of protein complexes formation involving P450 and its redox partner or other P450 enzymes. Finally, covalent immobilization on polymer surfaces or electrodes has benefited the areas of biocatalysis and biosensor design. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
[Mh] Termos MeSH primário: Reagentes para Ligações Cruzadas/química
Sistema Enzimático do Citocromo P-450/química
Proteínas Imobilizadas/química
Marcadores de Fotoafinidade/química
Coloração e Rotulagem/métodos
[Mh] Termos MeSH secundário: Acetilação
Regulação Alostérica
Biocatálise
Técnicas Biossensoriais
Domínio Catalítico
Sistema Enzimático do Citocromo P-450/metabolismo
Corantes Fluorescentes/química
Seres Humanos
Proteínas Imobilizadas/metabolismo
Isoenzimas/química
Isoenzimas/metabolismo
Modelos Moleculares
Oxirredução
Estrutura Secundária de Proteína
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; REVIEW
[Nm] Nome de substância:
0 (Cross-Linking Reagents); 0 (Fluorescent Dyes); 0 (Immobilized Proteins); 0 (Isoenzymes); 0 (Photoaffinity Labels); 9035-51-2 (Cytochrome P-450 Enzyme System)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180208
[Lr] Data última revisão:
180208
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170620
[St] Status:MEDLINE


  5 / 1915 MEDLINE  
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[PMID]:29025665
[Au] Autor:Adibi-Motlagh B; Lotfi AS; Rezaei A; Hashemi E
[Ad] Endereço:Department of Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.
[Ti] Título:Cell attachment evaluation of the immobilized bioactive peptide on a nanographene oxide composite.
[So] Source:Mater Sci Eng C Mater Biol Appl;82:323-329, 2018 Jan 01.
[Is] ISSN:1873-0191
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The immobilization of bioactive peptides as key molecules in numerous biological and physiological functions holds promise for designing advanced biomaterials. Graphene and its derivatives, having unique physicochemical properties, have brought considerable attention in the life sciences. In this regard, the chemical manipulation of the graphene surface with bioactive peptides opens a new horizon to design bioactive materials for a variety of future nanobiotechnologies. In this study, the first straightforward strategy for the covalent immobilization of the cell-adhesion peptide onto the graphene surface based on the Ugi four-component assembly process (Ugi 4-CAP) will be presented. The modified adhesion motif peptide, as an amine component in the presence of formaldehyde, cyclohexylisocyanide and carboxylated-graphene (G-COOH), was adopted in a four component reaction to fabricate a peptide-graphene (Peptide-G) biomaterial in water as a green solvent at an ambient temperature. The amino functional groups corresponded to the modified adhesion motif peptide and were immobilized onto the graphene sheets, which were quantified by the Kaiser test. The sheets were characterized by further analyses with FT-IR, AFM, UV-vis, Raman and thermogravimetric analyses. The Peptide-G biomaterial showed excellent biocompatibility. In addition, the Peptide-G treated surface, due to the presence of RGD on the surface of the graphene, significantly accelerated the proliferation of human mesenchymal stem cells (hMSCs) at a better rate regarding the tissue plate.
[Mh] Termos MeSH primário: Materiais Biocompatíveis/química
Grafite/química
Nanopartículas/química
Oligopeptídeos/química
[Mh] Termos MeSH secundário: Materiais Biocompatíveis/farmacologia
Células da Medula Óssea/citologia
Adesão Celular/efeitos dos fármacos
Proliferação Celular/efeitos dos fármacos
Células Cultivadas
Seres Humanos
Proteínas Imobilizadas/química
Proteínas Imobilizadas/farmacologia
Células Mesenquimais Estromais/citologia
Células Mesenquimais Estromais/efeitos dos fármacos
Células Mesenquimais Estromais/metabolismo
Microscopia de Força Atômica
Óxidos/química
Espectroscopia de Infravermelho com Transformada de Fourier
Termogravimetria
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Biocompatible Materials); 0 (Immobilized Proteins); 0 (Oligopeptides); 0 (Oxides); 7782-42-5 (Graphite); 78VO7F77PN (arginyl-glycyl-aspartic acid)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180205
[Lr] Data última revisão:
180205
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171014
[St] Status:MEDLINE


  6 / 1915 MEDLINE  
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[PMID]:29216587
[Au] Autor:Voitechovic E; Korepanov A; Kirsanov D; Legin A
[Ad] Endereço:St. Petersburg State University, St. Petersburg, Russia; Institute of Microelectronics of Barcelona, Barcelona, Spain. Electronic address: voitechovic.edita@gmail.com.
[Ti] Título:Quantification of immobilized protein in pharmaceutical production by bio-assisted potentiometric multisensor system.
[So] Source:J Pharm Biomed Anal;150:67-71, 2018 Feb 20.
[Is] ISSN:1873-264X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system. Surface-immobilized recombinant protein A from Staphylococcus aureus (SpA, expressed in Escherichia coli), which is commonly used as affinity ligand immobilized to stationary phase (сhromatography media) for monoclonal antibody purification was employed as the model object. Chromatography media samples containing different amounts of immobilized SpA were analyzed. Proteinase K from Tritirachium album was employed as a bio-transducer. We demonstrated that the suggested approach provides information about immobilized SpA concentration with 0.8mg/ml accuracy in the range 1-6.7mg/ml and within just 16min. Moreover, the proposed procedure requires no expensive materials and equipment and no bio-transducer immobilization. This method has potential of application for fast monitoring of other immobilized proteins in different tasks.
[Mh] Termos MeSH primário: Ensaio de Imunoadsorção Enzimática/métodos
Proteínas Imobilizadas/química
Potenciometria/métodos
Proteína Estafilocócica A/química
[Mh] Termos MeSH secundário: Endopeptidase K/química
Proteínas Imobilizadas/análise
Proteínas Recombinantes/análise
Proteínas Recombinantes/química
Reprodutibilidade dos Testes
Proteína Estafilocócica A/análise
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Immobilized Proteins); 0 (Recombinant Proteins); 0 (Staphylococcal Protein A); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180130
[Lr] Data última revisão:
180130
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171208
[St] Status:MEDLINE


  7 / 1915 MEDLINE  
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[PMID]:29235322
[Au] Autor:Bobrovnik SA; Demchenko MO; Komisarenko SV
[Ti] Título:Kinetic parameters of polyreactive immunoglobulins interaction with antigens in the presence of protamine.
[So] Source:Ukr Biochem J;88(3):29-35, 2016 May-Jun.
[Is] ISSN:2409-4943
[Cp] País de publicação:Ukraine
[La] Idioma:eng
[Ab] Resumo:The discovered earlier phenomenon of the enhancment of polyreactive immunoglobulines (PRIGs) binding to antigens in the presence of protamine and Tween 20 was investigated in more details. The comparative analysis of PRIGs reaction dynamics with immobilized antigen was provided. In addition, the rate constants for the reaction and the affinity constants of PRIGs-antigen binding in the presence or absence of optimal protamine concentration were determined. The rate constant of PRIGs-antigen reaction did not increase in the presence of protamine optimal concentration and was even reduced approximately twice. However, in the presence of protamine the concentration of reactive PRIGs molecules, that were able to interact with antigen, increased approximately 30 times, and this led to strong reaction rate increase. Protamine also influenced the affinity constant of PRIGs-antigen binding, which increased approximately three times. The suggestion was made that such protamine effect was due to its influence on the PRIGs molecules special structure, and, as a result of the conformational change PRIGs became able to bind more effectively to the antigens.
[Mh] Termos MeSH primário: Reações Antígeno-Anticorpo
Antígenos/química
Imunoglobulinas/química
Protaminas/química
Soroalbumina Bovina/química
[Mh] Termos MeSH secundário: Animais
Afinidade de Anticorpos
Especificidade de Anticorpos
Antígenos/imunologia
Bovinos
Ensaio de Imunoadsorção Enzimática
Proteínas Imobilizadas
Cinética
Camundongos
Polissorbatos/química
Salmão
Soroalbumina Bovina/imunologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antigens); 0 (Immobilized Proteins); 0 (Immunoglobulins); 0 (Polysorbates); 0 (Protamines); 27432CM55Q (Serum Albumin, Bovine)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180116
[Lr] Data última revisão:
180116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171214
[St] Status:MEDLINE
[do] DOI:10.15407/ubj88.03.029


  8 / 1915 MEDLINE  
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[PMID]:28742329
[Au] Autor:Shi B; Deng Y; Zhao P; Li X
[Ad] Endereço:Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School , 2199 Lishui Road West, Shenzhen 518055, China.
[Ti] Título:Selecting a DNA-Encoded Chemical Library against Non-immobilized Proteins Using a "Ligate-Cross-Link-Purify" Strategy.
[So] Source:Bioconjug Chem;28(9):2293-2301, 2017 09 20.
[Is] ISSN:1520-4812
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:DNA-encoded chemical libraries (DELs) have recently emerged and become an important technology platform in biomedical research and drug discovery. DELs containing large numbers of compounds can be prepared and selected against biological targets to discover novel ligands and inhibitors. In practice, DELs are usually selected against purified and immobilized proteins using the typical "bind-wash-elute" protocol; however, selection methods compatible with non-immobilized proteins would be able to greatly expand the target scope of DELs beyond purified proteins to more-complex and biologically relevant targets. Using a novel "ligate-cross-link-purify" strategy, we report here a method capable of selecting DELs against unmodified and non-immobilized protein targets. In addition, this method has shown excellent capability in identifying binders with moderate and weak affinities.
[Mh] Termos MeSH primário: Reagentes para Ligações Cruzadas/química
DNA/química
Descoberta de Drogas/métodos
Proteínas/metabolismo
Bibliotecas de Moléculas Pequenas/química
Bibliotecas de Moléculas Pequenas/farmacologia
[Mh] Termos MeSH secundário: Reagentes para Ligações Cruzadas/isolamento & purificação
DNA/isolamento & purificação
Seres Humanos
Proteínas Imobilizadas/metabolismo
Ligantes
Compostos Macrocíclicos/química
Compostos Macrocíclicos/isolamento & purificação
Compostos Macrocíclicos/farmacologia
Ligação Proteica
Bibliotecas de Moléculas Pequenas/isolamento & purificação
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cross-Linking Reagents); 0 (Immobilized Proteins); 0 (Ligands); 0 (Macrocyclic Compounds); 0 (Proteins); 0 (Small Molecule Libraries); 9007-49-2 (DNA)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171219
[Lr] Data última revisão:
171219
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170726
[St] Status:MEDLINE
[do] DOI:10.1021/acs.bioconjchem.7b00343


  9 / 1915 MEDLINE  
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[PMID]:28710276
[Au] Autor:Qasem-Abdullah H; Perach M; Livnat-Levanon N; Lewinson O
[Ad] Endereço:From the Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa 31096, Israel.
[Ti] Título:ATP binding and hydrolysis disrupt the high-affinity interaction between the heme ABC transporter HmuUV and its cognate substrate-binding protein.
[So] Source:J Biol Chem;292(35):14617-14624, 2017 Sep 01.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, vitamin B , heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism.
[Mh] Termos MeSH primário: Transportadores de Cassetes de Ligação de ATP/metabolismo
Trifosfato de Adenosina/metabolismo
Proteínas de Bactérias/metabolismo
Proteínas de Transporte/metabolismo
Heme/metabolismo
Hemeproteínas/metabolismo
Modelos Moleculares
Yersinia pestis/metabolismo
[Mh] Termos MeSH secundário: Transportadores de Cassetes de Ligação de ATP/química
Transportadores de Cassetes de Ligação de ATP/genética
Trifosfato de Adenosina/química
Apoenzimas/química
Apoenzimas/genética
Apoenzimas/metabolismo
Proteínas de Bactérias/química
Proteínas de Bactérias/genética
Proteínas de Transporte/química
Proteínas de Transporte/genética
Membrana Celular/química
Membrana Celular/metabolismo
Dimerização
Heme/química
Hemeproteínas/química
Hemeproteínas/genética
Holoenzimas/química
Holoenzimas/genética
Holoenzimas/metabolismo
Hidrólise
Proteínas Imobilizadas/química
Proteínas Imobilizadas/genética
Proteínas Imobilizadas/metabolismo
Cinética
Simulação de Acoplamento Molecular
Domínios e Motivos de Interação entre Proteínas
Multimerização Proteica
Receptores de Superfície Celular/química
Receptores de Superfície Celular/genética
Receptores de Superfície Celular/metabolismo
Proteínas Recombinantes
Ressonância de Plasmônio de Superfície
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Apoenzymes); 0 (Bacterial Proteins); 0 (Carrier Proteins); 0 (Hemeproteins); 0 (Holoenzymes); 0 (Immobilized Proteins); 0 (Receptors, Cell Surface); 0 (Recombinant Proteins); 0 (heme-binding protein); 42VZT0U6YR (Heme); 8L70Q75FXE (Adenosine Triphosphate)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170926
[Lr] Data última revisão:
170926
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170716
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M117.779975


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[PMID]:28686585
[Au] Autor:Lowndes M; Junyent S; Habib SJ
[Ad] Endereço:Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK.
[Ti] Título:Constructing cellular niche properties by localized presentation of Wnt proteins on synthetic surfaces.
[So] Source:Nat Protoc;12(7):1498-1512, 2017 Jul.
[Is] ISSN:1750-2799
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Wnt signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the Wnt pathway can promote stem cell self-renewal and/or direct lineage commitment. Wnt proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing Wnt signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of Wnt proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces. Here we describe a detailed protocol based on covalent binding of nucleophilic groups on Wnt proteins to activated carboxylic acid (COOH) or glutaraldehyde (COH) groups functionalized on synthetic surfaces. As an example, we describe how this method can be used to covalently immobilize Wnt3a proteins on microbeads or a glass surface. This procedure requires ∼3 h and allows for the hydrophobic protein to be stored in the absence of detergent. The immobilization efficiency of active Wnt proteins can be assessed using different T-cell factor (TCF) reporter assays as a readout for Wnt/ß-catenin-dependent transcription. Immobilization efficiency can be measured 12-18 h after seeding the cells and takes 2-4 h. The covalent immobilization of Wnt proteins can also be used for single-cell analysis using Wnt-coated microbeads (12-18 h of live imaging) and to create a Wnt platform on a glass surface for stem cell maintenance and cell population analysis (3 d). The simple chemistry used for Wnt immobilization allows for adaptation to new materials and other developmental signals. Therefore, this method can also be incorporated into tissue engineering platforms in which depletion of the stem cell pool restricts the complexity and maturity of the tissue developed.
[Mh] Termos MeSH primário: Técnicas Citológicas/métodos
Proteínas Imobilizadas/metabolismo
Proteínas Wnt/metabolismo
[Mh] Termos MeSH secundário: Vidro
Microesferas
Ligação Proteica
Análise de Célula Única/métodos
Fatores de Tempo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Immobilized Proteins); 0 (Wnt Proteins)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170726
[Lr] Data última revisão:
170726
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170708
[St] Status:MEDLINE
[do] DOI:10.1038/nprot.2017.061



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