Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Methods Mol Biol ; 2127: 373-396, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32112334

RESUMO

NMR spectroscopy is a method of choice to characterize structure, function, and dynamics of integral membrane proteins at atomic resolution. Here, we describe protocols for sample preparation and characterization by NMR spectroscopy of two integral membrane proteins with different architecture, the α-helical membrane protein MsbA and the ß-barrel membrane protein BamA. The protocols describe recombinant expression in E. coli, protein refolding, purification, and reconstitution in suitable membrane mimetics, as well as key setup steps for basic NMR experiments. These include experiments on protein samples in the solid state under magic angle spinning (MAS) conditions and experiments on protein samples in aqueous solution. Since MsbA and BamA are typical examples of their respective architectural classes, the protocols presented here can also serve as a reference for other integral membrane proteins.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Lasers de Estado Sólido , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/instrumentação , Proteínas de Membrana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Renaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
3.
Nature ; 576(7787): 452-458, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31645764

RESUMO

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Animais , Antibacterianos/efeitos adversos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Produtos Biológicos/química , Descoberta de Drogas , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fluorescência , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/patogenicidade , Humanos , Lipopolissacarídeos/química , Compostos Macrocíclicos/efeitos adversos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutação , Peptidomiméticos/efeitos adversos , Marcadores de Fotoafinidade
4.
J Biomol NMR ; 73(6-7): 375-384, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31073665

RESUMO

The insertase BamA is an essential protein of the bacterial outer membrane. Its 16-stranded transmembrane ß-barrel contains a lateral gate as a key functional element. This gate is formed by the C-terminal half of the last ß-strand. The BamA barrel was previously found to sample different conformations in aqueous solution, as well as different gate-open, gate-closed, and collapsed conformations in X-ray crystallography and cryo-electron microscopy structures. Here, we report the successful identification of conformation-selective nanobodies that stabilize BamA in specific conformations. While the initial candidate generation and selection protocol was based on established alpaca immunization and phage display selection procedures, the final selection of nanobodies was enhanced by a solution NMR-based screening step to shortlist the targets for crystallization. In this way, three crystal structures of BamA-nanobody complexes were efficiently obtained, showing two types of nanobodies that indeed stabilized BamA in two different conformations, i.e., with open and closed lateral gate, respectively. Then, by correlating the structural data with high resolution NMR spectra, we could for the first time assign the BamA conformational solution ensemble to defined structural states. The new nanobodies will be valuable tools towards understanding the client insertion mechanism of BamA and towards developing improved antibiotics.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Modelos Moleculares , Conformação Proteica , Anticorpos de Domínio Único/química , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Anticorpos de Domínio Único/farmacologia , Soluções
5.
J Am Chem Soc ; 140(36): 11252-11260, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30125090

RESUMO

ß-barrel outer membrane proteins (Omps) are key functional components of the outer membranes of Gram-negative bacteria, mitochondria, and plastids. In bacteria, their biogenesis requires the ß-barrel-assembly machinery (Bam) with the central insertase BamA, but the exact translocation and insertion mechanism remains elusive. The BamA insertase features a loosely closed gating region between the first and last ß-strand 16. Here, we describe ∼70% complete sequence-specific NMR resonance assignments of the transmembrane region of the BamA ß-barrel in detergent micelles. On the basis of the assignments, NMR spectra show that the BamA barrel populates a conformational ensemble in slow exchange equilibrium, both in detergent micelles and lipid bilayer nanodiscs. Individual conformers can be selected from the ensemble by the introduction of a C-terminal strand extension, single-point mutations, or specific disulfide cross-linkings, and these modifications at the barrel seam are found to be allosterically coupled to sites at the entire barrel circumference. The resonance assignment provides a platform for mechanistic studies of BamA at atomic resolution, as well as for investigating interactions with potential antibiotic drugs and partner proteins.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...