RESUMO
NMR diffusometry has been gaining wide popularity in various areas of applied chemistry for investigating diffusion and complexation processes in solid and aqueous phases. To date, the application of this method to study aggregation phenomena proceeding beyond the dimer stage of assembly has been restricted by the need for a priori knowledge of the aggregates' shape, commonly difficult to know in practice. We describe here a comprehensive analysis of aggregation parameter-dependency on the type and shape selected for modeling assembly processes, and report for the first time a shape-independent model (designated the SHIM approach), which may be used as an alternative in cases when information on aggregates' shapes is unavailable. The model can be used for determining equilibrium aggregation parameters from self-diffusion NMR data including equilibrium self-association constant and changes in enthalpy, ΔH, and entropy, ΔS.
Assuntos
Espectroscopia de Ressonância Magnética , Modelos Moleculares , Complexos de Coordenação/química , Difusão , Hidrodinâmica , TermodinâmicaRESUMO
Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo cross-linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia coli cells. We identified two signal-sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA).
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cisteína/metabolismo , Histidina Quinase , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Transdução de SinaisRESUMO
Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In Escherichia coli, chemoreceptors exhibit higher-order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer-of-dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo cross-linking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr, respectively. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed cross-links, whereas reporters located different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernible effect on the cross-linking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced cross-linking at most of the reporter sites, indicating that individual dimers may move closer together under this condition.
Assuntos
Proteínas de Bactérias/química , Células Quimiorreceptoras/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Multimerização Proteica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Quimiorreceptoras/metabolismo , Reagentes de Ligações Cruzadas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície CelularRESUMO
During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.
Assuntos
Quimiotaxia , Proteínas de Escherichia coli/biossíntese , Escherichia coli/fisiologia , Expressão Gênica , Transdução de Sinais , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos BiológicosRESUMO
The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H(+)-treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 - EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (K(I) (*)) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection.
Assuntos
Echinococcus granulosus/metabolismo , Serina Proteases/química , Sequência de Aminoácidos , Animais , Cães , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Genes de Helmintos , Interações Hospedeiro-Parasita , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pepsina A/química , Inibidores de Proteases/química , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina Proteases/metabolismoRESUMO
The chemoreceptor molecules that mediate chemotactic responses in bacteria and archaea are physically clustered and operate as highly cooperative arrays. Few experimental approaches are able to investigate the structure-function organization of these chemoreceptor networks in living cells. This chapter describes chemical crosslinking methods that can be applied under normal physiological conditions to explore physical interactions between chemoreceptors and their underlying genetic and structural basis. Most of these crosslinking approaches are based on available atomic structures for chemoreceptor homodimers, the fundamental building block for higher-order networks. However, the general logic of our in vivo crosslinking approaches is readily applicable to other protein-protein interactions and other organisms, even when high-resolution structural information is not available.
Assuntos
Bioquímica/métodos , Células Quimiorreceptoras/química , Reagentes de Ligações Cruzadas/farmacologia , Proteínas Arqueais/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Cisteína/química , Citoplasma/metabolismo , Dimerização , Dissulfetos/química , Proteínas de Escherichia coli/química , Lisina/química , Modelos Químicos , Modelos Genéticos , Conformação Molecular , Mutação , Ligação ProteicaRESUMO
BACKGROUND: Parasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity. RESULTS: Taking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda. CONCLUSIONS: These results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.