Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 121(39): e2413100121, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39292747

RESUMO

The adenosine di-phosphate (ADP) ribosylation factor (Arf) small guanosine tri-phosphate (GTP)ases function as molecular switches to activate signaling cascades that control membrane organization in eukaryotic cells. In Arf1, the GDP/GTP switch does not occur spontaneously but requires guanine nucleotide exchange factors (GEFs) and membranes. Exchange involves massive conformational changes, including disruption of the core ß-sheet. The mechanisms by which this energetically costly switch occurs remain to be elucidated. To probe the switch mechanism, we coupled pressure perturbation with nuclear magnetic resonance (NMR), Fourier Transform infra-red spectroscopy (FTIR), small-angle X-ray scattering (SAXS), fluorescence, and computation. Pressure induced the formation of a classical molten globule (MG) ensemble. Pressure also favored the GDP to GTP transition, providing strong support for the notion that the MG ensemble plays a functional role in the nucleotide switch. We propose that the MG ensemble allows for switching without the requirement for complete unfolding and may be recognized by GEFs. An MG-based switching mechanism could constitute a pervasive feature in Arfs and Arf-like GTPases, and more generally, the evolutionarily related (Ras-like small GTPases) Rags and Gα GTPases.


Assuntos
Fator 1 de Ribosilação do ADP , Guanosina Difosfato , Guanosina Trifosfato , Guanosina Difosfato/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Guanosina Trifosfato/metabolismo , Humanos , Espalhamento a Baixo Ângulo , Difração de Raios X , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Modelos Moleculares
2.
Biochim Biophys Acta Bioenerg ; 1862(8): 148432, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33932367

RESUMO

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) is a main ion transporter in many pathogenic bacteria. We previously proposed that N-terminal stretch of the NqrB subunit plays an important role in regulating the ubiquinone reaction at the adjacent NqrA subunit in Vibrio cholerae Na+-NQR. However, since approximately three quarters of the stretch (NqrB-Met1-Pro37) was not modeled in an earlier crystallographic study, its structure and function remain unknown. If we can develop a method that enables pinpoint modification of this stretch by functional chemicals (such as spin probes), it could lead to new ways to investigate the unsettled issues. As the first step to this end, we undertook to specifically attach an alkyne group to a lysine located in the stretch via protein-ligand affinity-driven substitution using synthetic ligands NAS-K1 and NAS-K2. The alkyne, once attached, can serve as an "anchor" for connecting functional chemicals via convenient click chemistry. After a short incubation of isolated Na+-NQR with these ligands, alkyne was predominantly incorporated into NqrB. Proteomic analyses in combination with mutagenesis of predicted target lysines revealed that alkyne attaches to NqrB-Lys22 located at the nonmodeled region of the stretch. This study not only achieved the specific modification initially aimed for but also provided valuable information about positioning of the nonmodeled region. For example, the fact that hydrophobic NAS-Ks come into contact with NqrB-Lys22 suggests that the nonmodeled region may orient toward the membrane phase rather than protruding into cytoplasmic medium. This conformation may be essential for regulating the ubiquinone reaction in the adjacent NqrA.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Lisina/química , Proteoma/análise , Vibrio cholerae/enzimologia , Sítios de Ligação , Transporte de Íons , Conformação Proteica , Subunidades Proteicas , Sódio/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(39): 24484-24493, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32938803

RESUMO

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active Bacteroides fragilis The ability to visualize fluorescently labeled Bacteroides and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.


Assuntos
Bacteroides/metabolismo , Microbioma Gastrointestinal , Aerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/classificação , Bacteroides/genética , Bacteroides/crescimento & desenvolvimento , Humanos , Oxigênio/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo
4.
mBio ; 11(1)2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019804

RESUMO

In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H+ or Na+). We know little about the respiratory pathways in Bacteroides species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut Bacteroides species, Bacteroides fragilis, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na+-pumping NADH:quinone oxidoreductase (NQR) and NADH dehydrogenase II (NDH2), have NADH dehydrogenase activity, whereas H+-pumping NADH:ubiquinone oxidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δnqr Δndh2 mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered in vitro growth, the single nqr deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in B. fragilis and the overall importance of respiration to this abundant gut symbiont.IMPORTANCEBacteroides species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of Bacteroides species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that B. fragilis has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in Bacteroides, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts.


Assuntos
Bacteroides fragilis/enzimologia , Bacteroides fragilis/genética , Anaerobiose , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Feminino , Vida Livre de Germes , Masculino , Redes e Vias Metabólicas , Camundongos , NAD/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Oxirredução , Quinona Redutases/genética , Quinona Redutases/metabolismo , Deleção de Sequência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA