RESUMO
Eukaryotic genomes are folded in a hierarchical organization that reflects and possibly regulates their function. Genomewide studies revealed a new level of organization at the kilobase-to-megabase scale termed "topological associating domains" (TADs). TADs are characterized as stable units of chromosome organization that restrict the action of regulatory sequences within one "functional unit." Consequently, TADs are expected to appear as physical entities in most cells. Very recent single-cell studies have shown a notable variability in genome architecture at this scale, raising concerns about this model. Furthermore, the direct and simultaneous observation of genome architecture and transcriptional output showed the lack of stable interactions between regulatory sequences in transcribing cells. These findings are consistent with a large body of evidence suggesting that genome organization is highly heterogeneous at different scales. In this review, we discuss the main strategies employed to image chromatin organization, present the latest state-of-the-art developments, and propose an interpretation reconciling population-based findings with direct single-cell chromatin organization observations. All in all, we propose that TADs are made of multiple, low-frequency, low-affinity interactions that increase the probability, but are not deterministic, of regulatory interactions.
Assuntos
Cromatina/química , Cromatina/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Conformação Molecular , Imagem Individual de Molécula , Eucariotos , MicroscopiaRESUMO
Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Trifosfato de Adenosina/metabolismo , Difusão , Hidrólise , Cinética , Microscopia de Força Atômica , Imagem Individual de Molécula , Esporos Bacterianos/metabolismoRESUMO
Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites.
Assuntos
Modelos Biológicos , Ligação Proteica , Sítios de Ligação , Metabolismo Energético , Cinética , Ligantes , Substâncias Macromoleculares/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: To evaluate the mechanisms of microbial interaction between the oral pathogens Candida albicans and Streptococcus mutans. MATERIALS AND METHODS: Growth kinetics for the two micro-organisms, cultured individually or together, were followed experimentally for 36 h. The different growth curves were analysed by means of mathematical modelling. RESULTS: Under the experimental conditions, S. mutans final concentration, when grown individually, was 5-times that of C. albicans. Contrarily, when both micro-organisms grew together, this ratio was inversed and C. albicans final concentration was even higher than that of S. mutans. When both micro-organisms share the niche, a model including linear competition among one another was best suited to reproduce the experimental observations. The results of this model show that the initial growth rates of both species are positively influenced by their mutual interaction. However, at longer incubation times, C. albicans prevents bacterial growth and achieves concentrations 4-times higher than when grown individually. CONCLUSIONS: The results suggest that C. albicans biofilm formation could be potentiated by the presence of S. mutans by two mechanisms: synergically at short times and by competition at longer periods.
Assuntos
Candida albicans/fisiologia , Modelos Teóricos , Streptococcus mutans/fisiologia , Candida albicans/crescimento & desenvolvimento , Streptococcus mutans/genéticaRESUMO
Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes.
Assuntos
Proteínas de Membrana/química , Modelos Moleculares , Fosfolipídeos/química , Algoritmos , Ativação Enzimática , Humanos , Proteínas de Membrana/metabolismo , Micelas , Fosfolipídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/químicaRESUMO
Although 1-anilino-naphthalene-8-sulfonate (ANS) has been widely used in protein folding and binding studies, the detailed mechanism of this interaction is not fully understood. In this work the binding of ANS was analyzed at pre-equilibrium and equilibrium conditions using bovine serum albumin (BSA) as model. We employed a combined approach including the analysis of fluorescence, near-UV circular dichroism and isothermal titration calorimetric data. Experiments at equilibrium with these techniques identify three ANS molecules bound at hydrophobic cavities in BSA. Pre-equilibrium fluorescence analysis unambiguously indicated that the binding of ANS at hydrophobic cavities of BSA occurs at two different and independent classes of sites with similar affinities and quantum yields, two features that are undetectable by the equilibrium analysis. The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (DeltaH = -22 kJ/mol) and entropic terms (-TDeltaS = -17 kJ/mol), while the binding to the second site is enthalpically driven (DeltaH = -31 kJ/mol; -TDeltaS = -0.6 kJ/mol). Complementary information from molecular docking showed three ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally and three additional ANS molecules bound at water exposed sites.
Assuntos
Naftalenossulfonato de Anilina/química , Soroalbumina Bovina/química , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.