RESUMEN
Single-stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 domains led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1's multi-domain organization. Here, we directly probe this process at the single-molecule level. Quantitative smFRET and structural ensemble calculations reveal how PARP-1's N-terminal zinc fingers convert DNA SSBs from a largely unperturbed conformation, via an intermediate state into the highly kinked DNA conformation. Our data suggest an induced fit mechanism via a multi-domain assembly cascade that drives SSB sensing and stimulates an interplay with the scaffold protein XRCC1 orchestrating subsequent DNA repair events. Interestingly, a clinically used PARP-1 inhibitor Niraparib shifts the equilibrium towards the unkinked DNA conformation, whereas the inhibitor EB47 stabilizes the kinked state.
Asunto(s)
Roturas del ADN de Cadena Simple , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Reparación del ADN , Daño del ADN , ADN/metabolismoRESUMEN
Förster resonance energy transfer (FRET) can be used to measure distances and infer structures at the molecular level. However, the flexible linkers with which the fluorophores are attached to a macromolecule introduce a lack of knowledge. Both the dye's geometry and kinetics give rise to uncertainties. Whereas the impact of the geometry is already well understood, the real extent of the kinetics has not been investigated thoroughly. Here, we present a single-molecule (sm)FRET theory that defines the kinetics of dye movements in a complete form. We introduce a formal nomenclature and provide a recipe for the calculation of the corresponding FRET efficiency. We further analyze experimental data in order to obtain parameters characterizing the geometry and kinetics of the FRET dyes and use them to resimulate the FRET efficiencies by diffusion of fluorophore and linker movement. We show in a real case scenario of dye molecules attached to dsDNA that when making geometrical and kinetic assumptions commonly used in the FRET community one obtains results differing from the experimental data. In contrast, our stochastic simulations taking kinetic parameters from experiments into account reproduce the correct FRET efficiencies. Furthermore, we present a method enabling us to classify the kinetics of the dyes by investigating single realizations of the simulated transfer process. The results support our notion that the common kinetic assumptions are not appropriate over the whole range of distances inferred by FRET even for the simple situation of dyes attached to DNA where few interactions occur.
Asunto(s)
ADN/química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Cinética , Simulación de Dinámica MolecularRESUMEN
This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.
RESUMEN
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Laboratorios/normas , Reproducibilidad de los ResultadosRESUMEN
Single-molecule Förster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment. Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Nanotecnología/métodos , Algoritmos , Anisotropía , Teorema de Bayes , Cristalografía por Rayos X , Fluorescencia , Método de MontecarloRESUMEN
The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca(2+) and regulation of Ca(2+)-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca(2+) release from intracellular stores; (iii) Ca(2+) entry from the extracellular compartment; and (iv) nuclear translocation of the Ca(2+)-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca(2+) signaling.
Asunto(s)
Señalización del Calcio/fisiología , Fosfolipasa C gamma/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Transporte Activo de Núcleo Celular , Sustitución de Aminoácidos , Animales , Proteínas Aviares/química , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular , Pollos , Humanos , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFATC/metabolismo , Fosfolipasa C gamma/química , Fosfolipasa C gamma/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rac/química , Proteínas de Unión al GTP rac/genéticaRESUMEN
Nanoparticle uptake by living cells is governed by chemical interactions between functional groups on the nanoparticle as well as the receptors on cell surfaces. Here we have investigated the uptake of anionic polystyrene (PS) nanoparticles of â¼100 nm diameter by mesenchymal stem cells (MSCs) using spinning-disk confocal optical microscopy combined with a quantitative analysis of the fluorescence images. Two types of anionic PS nanoparticles with essentially identical sizes and ζ-potentials were employed in this study, carboxyl-functionalized nanoparticles (CPS) and plain PS nanoparticles, both coated with anionic detergent for stabilization. CPS nanoparticles were observed to internalize more rapidly and accumulate to a much higher level than plain PS nanoparticles. The relative importance of different uptake mechanisms for the two types of nanoparticles was investigated by using specific inhibitors. CPS nanoparticles were internalized mainly via the clathrin-mediated mechanism, whereas plain PS nanoparticles mainly utilized the macropinocytosis pathway. The pronounced difference in the internalization behavior of CPS and plain PS nanoparticles points to a specific interaction of the carboxyl group with receptors on the cell surface.
Asunto(s)
Células Madre Mesenquimatosas/química , Microscopía Fluorescente/métodos , Nanopartículas/química , Nanopartículas/ultraestructura , Poliestirenos/química , Aniones , Células Cultivadas , Humanos , Ensayo de Materiales , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Although systemically applied nanoparticles are quickly taken up by phagocytic cells, mainly macrophages, the interactions between engineered nanoparticles and macrophages are still not well defined. We therefore analyzed the uptake of diagnostically used carboxydextran-coated superparamagnetic iron oxide nanoparticles of 60 nm (SPIO) and 20 nm (USPIO) by human macrophages. By pharmacological and in vitro knockdown approaches, the principal uptake mechanism for both particles was identified as clathrin-mediated, scavenger receptor A-dependent endocytosis. We developed a mathematical model of the uptake process that allows determination of key parameters of endocytosis, including the rate of uptake, the number of nanoparticles per cell in saturation, the mean uptake time, and the correlation between the number of internalized nanoparticles and their extracellular concentration. The calculated parameters correlate well with experimental data obtained by confocal microscopy. Moreover, the model predicts the individual and collective wrapping times of different nanoparticles, describes the relation between cytoskeletal forces, membrane elasticity and the uptake time. We also introduced a new physical parameter 'a' governing the collective uptake process, a reflecting minimal linear spacing between simultaneously acting neighboring endocytotic pits.
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Endocitosis/fisiología , Compuestos Férricos/química , Macrófagos/metabolismo , Nanopartículas/química , Polímeros/química , Células Cultivadas , Humanos , Macrófagos/ultraestructura , Imagen por Resonancia Magnética , Microscopía Confocal , Microscopía Electrónica de TransmisiónRESUMEN
Colloidal nanoparticles are often stabilized by high surface charges. These create an electrical potential that may strongly affect the concentration of dissolved ions, which presents a formidable problem for the use of nanoparticles in ion-sensing applications. This effect is investigated systematically with organic fluorophore-gold nanoparticle hybrids, which have a chloride-sensitive fluorophore attached at varying distances from their surface. The distance-dependent fluorescence response is quantitatively assessed using fluorescence spectroscopy.
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Nanopartículas/química , Nanotecnología/métodos , Cloruros/química , Espectrometría de FluorescenciaRESUMEN
Uptake and intracellular transport of D-penicillamine coated quantum dots (DPA-QDs) of 4 nm radius by live HeLa cells have been investigated systematically by spinning disk and 4Pi confocal microscopies. Unlike larger nanoparticles, these small DPA-QDs were observed to accumulate at the plasma membrane prior to internalization, and the uptake efficiency scaled nonlinearly with the nanoparticle concentration. Both observations indicate that a critical threshold density has to be exceeded for triggering the internalization process. By using specific inhibitors, we showed that DPA-QDs were predominantly internalized by clathrin-mediated endocytosis and to a smaller extent by macropinocytosis. Clusters of DPA-QDs were found in endosomes, which were actively transported along microtubules toward the perinuclear region. Later on, a significant fraction of endocytosed DPA-QDs were found in lysosomes, while others were actively transported to the cell periphery and exocytosed with a half-life of 21 min.
Asunto(s)
Endocitosis , Exocitosis , Penicilamina/química , Penicilamina/metabolismo , Puntos Cuánticos , Tampones (Química) , Membrana Celular/metabolismo , Supervivencia Celular , Células HeLa , Humanos , Cinética , Microscopía ConfocalRESUMEN
Contrast agents based on dextran-coated superparamagnetic iron oxide nanoparticles (SPIO) are internalized by professional phagocytes such as hepatic Kupffer cells, yet their role in phagocyte biology remains largely unknown. Here we investigated the effects of the SPIO ferucarbotran on murine Kupffer cells and human macrophages. Intravenous injection of ferucarbotran into mice led to rapid accumulation of the particles in phagocytes and to long-lasting increased iron deposition in liver and kidneys. Macrophages incorporate ferucarbotran in lysosomal vesicles containing α-glucosidase, which is capable of degrading the carboxydextran shell of the ferucarbotran particles. Intravenous injection of ferucarbotran into mice followed by incorporation of the nanoparticles into Kupffer cells triggered apoptosis and the subsequent depletion of Kupffer cells. In macrophages, the proinflammatory cytokine TNF-α increased the apoptosis rate, the reactive oxygen species production and the activation of c-Jun N-terminal kinase elicited by ferucarbotran, which might be mediated by the induction of cytoplasmic phospholipase A2 by TNF-α. Notably, the nanoparticle-induced apoptosis of murine Kupffer cells could be prevented by treatment of the mice with the radical scavenger edaravone. Thus, nanosized carboxydextran-coated SPIO-based contrast agents are retained for extended time periods by liver macrophages, where they elicit delayed cell death, which can be antagonized by a therapeutic radical scavenger.
Asunto(s)
Dextranos/metabolismo , Lisosomas/metabolismo , Nanopartículas/química , Fagocitos/metabolismo , Animales , Antipirina/análogos & derivados , Antipirina/farmacología , Apoptosis/efectos de los fármacos , Dextranos/farmacocinética , Edaravona , Depuradores de Radicales Libres/farmacología , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Cinética , Macrófagos del Hígado/citología , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Lisosomas/efectos de los fármacos , Imagen por Resonancia Magnética , Nanopartículas de Magnetita , Ratones , Fagocitos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Superparamagnetic iron oxide nanoparticles are frequently used for cell labeling or as diagnostic contrast media, yet studies analyzing their effects on immune cells remain scarce. Here we investigated how nanosized carboxydextran-coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) might affect human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages. Confocal microscopy revealed that after 24 h the particles were almost exclusively localized within the lysosomal compartment. Continued cultivation of the macrophages for several days was associated with apoptosis induction caused by a long-lasting activation of the c-Jun N-terminal kinase (JNK) pathway. JNK activation was due to significantly elevated levels of reactive oxygen species, whereas no TNF-alpha was produced by the macrophages treated with nanoparticles. Compared to SPIO, USPIO induced more pronounced biochemical alterations and cytotoxicity, which could be antagonized by the JNK inhibitor V. Alternatively, treatment of macrophages with Trolox or N-acetyl-L-cysteine, two functionally different scavengers of reactive oxygen species, abolished both the JNK activation and the subsequent cytotoxic effects. These data indicate that nanosized superparamagnetic iron oxide-based contrast media exert cytotoxicity in human macrophages that can be functionally antagonized with radical scavengers.
Asunto(s)
Apoptosis/fisiología , Materiales Biocompatibles Revestidos/administración & dosificación , Dextranos/administración & dosificación , Óxido Ferrosoférrico/administración & dosificación , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/citología , Macrófagos/enzimología , Nanopartículas/administración & dosificación , Apoptosis/efectos de los fármacos , Células Cultivadas , Humanos , Macrófagos/efectos de los fármacos , Nanopartículas de MagnetitaRESUMEN
Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.
Asunto(s)
Inflamación/patología , Espacio Intracelular/metabolismo , Pulmón/metabolismo , Pulmón/patología , Magnetismo/métodos , Nanopartículas del Metal/química , Bioensayo , Transporte Biológico , Barrera Alveolocapilar/metabolismo , Barrera Alveolocapilar/patología , Agregación Celular , Células Cultivadas , Fluorescencia , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Inflamación/metabolismo , Hierro/química , Lisosomas/metabolismo , Nanopartículas del Metal/ultraestructura , Microscopía Confocal , Modelos Biológicos , Tamaño de la Partícula , Platino (Metal)/química , Ácidos Polimetacrílicos/químicaRESUMEN
We have investigated the uptake of cationic polystyrene nanoparticles by mesenchymal stem cells (MSCs) using confocal fluorescence microscopy and flow cytometry. Two types of nanoparticles of about 100 nm diameter with similar zeta potentials were employed in this study, plain polystyrene (PS) nanoparticles and amino-functionalized polystyrene (NPS) nanoparticles, each carrying about 6000 amino groups on the surface. To assess the relative importance of specific endocytosis mechanisms, uptake was observed in the presence of the drugs dynasore and chlorpromazine. NPS nanoparticles were rapidly internalized and accumulated to a much higher level in MSCs than PS nanoparticles, predominantly via the main clathrin-mediated pathway. PS nanoparticles were internalized mainly via clathrin-independent endocytosis. The pronounced difference in the internalization behavior of PS and NPS nanoparticles points to specific interactions of the amino groups on the nanoparticle surface with the endocytosis machinery of the cells.
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Aminas/química , Células Madre Mesenquimatosas/química , Nanopartículas , Poliestirenos/química , Células Cultivadas , Citometría de Flujo , Humanos , Células Madre Mesenquimatosas/citología , Microscopía ConfocalRESUMEN
Nanoparticles are finding a rapidly expanding range of applications in research and technology, finally entering our daily life in medical, cosmetic or food products. Their ability to invade all regions of an organism including cells and cellular organelles offers new strategies for medical diagnosis and therapy (nanomedicine), but their safe use requires a deep knowledge about their interactions with biological systems at the molecular level. Upon incorporation, nanoparticles are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a 'protein corona'. These nanoparticle-protein interactions are still poorly understood and quantitative studies to characterize them remain scarce. Here we have quantitatively analysed the adsorption of human transferrin onto small (radius approx. 5 nm) polymer-coated FePt nanoparticles by using fluorescence correlation spectroscopy. Transferrin binds to the negatively charged nanoparticles with an affinity of approximately 26 microM in a cooperative fashion and forms a monolayer with a thickness of 7 nm. By using confocal fluorescence microscopy, we have observed that the uptake of FePt nanoparticles by HeLa cells is suppressed by the protein corona compared with the bare nanoparticles.
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Modelos Moleculares , Nanopartículas/química , Transferrina/química , Adsorción , Células HeLa , Humanos , Hierro/química , Microscopía Confocal , Platino (Metal)/química , Espectrometría de FluorescenciaRESUMEN
It is now known that nanoparticles, when exposed to biological fluid, become coated with proteins and other biomolecules to form a 'protein corona'. Recent systematic studies have identified various proteins that can make up this corona, but these nanoparticle-protein interactions are still poorly understood, and quantitative studies to characterize them are few in number. Here, we have quantitatively analysed the adsorption of human serum albumin onto small (10-20 nm in diameter) polymer-coated FePt and CdSe/ZnS nanoparticles by using fluorescence correlation spectroscopy. The protein corona forms a monolayer with a thickness of 3.3 nm. Proteins bind to the negatively charged nanoparticles with micromolar affinity, and time-resolved fluorescence quenching experiments show that they reside on the particle for approximately 100 s. These new findings deepen our quantitative understanding of the protein corona, which is of utmost importance in the safe application of nanoscale objects in living organisms.
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Nanopartículas/análisis , Nanopartículas/química , Mapeo de Interacción de Proteínas/métodos , Puntos Cuánticos , Albúmina Sérica/química , Espectrometría de Fluorescencia/métodos , Fenómenos Fisiológicos Celulares , Humanos , Ensayo de MaterialesRESUMEN
A monomeric variant of the red fluorescent protein eqFP611, mRuby, is described. With excitation and emission maxima at 558 nm and 605 nm, respectively, and a large Stokes shift of 47 nm, mRuby appears particularly useful for imaging applications. The protein shows an exceptional resistance to denaturation at pH extremes. Moreover, mRuby is about ten-fold brighter compared to EGFP when being targeted to the endoplasmic reticulum. The engineering process of eqFP611 revealed that the C-terminal tail of the protein acts as a natural peroxisomal targeting signal (PTS). Using an mRuby variant carrying the eqFP611-PTS, we discovered that ordered inheritance of peroxisomes is widespread during mitosis of different mammalian cell types. The ordered partitioning is realized by the formation of peroxisome clusters around the poles of the mitotic spindle and ensures that equal numbers of the organelle are inherited by the daughter cells. The unique spectral properties make mRuby the marker of choice for a multitude of cell biological applications. Moreover, the use of mRuby has allowed novel insights in the biology of organelles responsible for severe human diseases.
Asunto(s)
Estructuras Celulares/metabolismo , Proteínas Luminiscentes/metabolismo , Coloración y Etiquetado , Animales , Línea Celular , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Peroxisomas/metabolismo , Proteína Fluorescente RojaRESUMEN
A molecular model of the acidic compact state of apomyoglobin (A-state) from yellowfin tuna was obtained using molecular dynamics simulations (MD) by calculating multiple trajectories. To cause partial unfolding within a reasonable amount of CPU time, both an acidic environment (pH 3 and 0.15M NaCl) and a temperature jump to 500 K were needed. Twenty-five acidic structures of apomyoglobin were generated by MD, 10 of them can be clustered by RMSD in an average structure having a common hydrophobic core as was reported for acidic sperm whale apomyoglobin, with shortened helices A,G,E, and H (the helix A appears to be translated along the sequence). Prolonging the MD runs at 500 K did not cause further substantial unfolding, suggesting that the ensemble of generated structures is indicative of a region of the conformational space accessible to the apoprotein at acidic pH corresponding to a local energy minimum. The comparison of experimentally determined values of specific spectroscopic properties of the apomyoglobin in acidic salt conditions with the expected ones on the basis of the MD generated structures shows a reasonable agreement considering the characteristic uncertainties of both experimental and simulation techniques. We used frequency domain fluorometry, acrylamide fluorescence quenching, and fluorescence correlation spectroscopy together with far UV circular dichroism to estimate the helical content, the Stern-Volmer quenching constant and the radius of gyration of the protein. Tuna apomyoglobin is a single tryptophan protein and thus, interpretation of its intrinsic fluorescence is simpler than for other proteins. The high sensitivity of the applied fluorescence techniques enabled experiments to be performed under very dilute conditions, that is, at concentrations of subnanomolar for the FCS measurements and 6 muM for the other fluorescence measurements. As high concentrations of proteins can strongly affect the association equilibrium among partially unfolded states, fluorescence techniques can provide complementary information with respect to other techniques requiring higher sample concentrations, such as NMR. The analysis of exposed hydrophobic regions in each of the MD-generated acidic structures reveals potential candidates involved in the aggregation processes of apomyoglobin in the acidic compact state. Our investigation represents an effective model system for studying amyloid fibril formation found in important diseases that are believed to proceed via aggregation of protein in the molten globule state.
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Apoproteínas/química , Proteínas de Peces/química , Mioglobina/química , Atún/metabolismo , Acrilamida/química , Animales , Dicroismo Circular , Simulación por Computador , Calor , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Cloruro de Sodio/química , Espectrometría de FluorescenciaRESUMEN
C-reactive protein (CRP) is a prototype acute-phase protein that may be intimately involved in human disease. Its cellular receptors are still under debate; the main candidates are FcR for immunoglobulin G, as CRP was shown to bind specifically to FcgammaRI and FcgammaRIIa. Using ultrasensitive confocal live-cell imaging, we have studied CRP binding to FcgammaR naturally expressed in the plasma membranes of cells from a human leukemia cell line (Mono Mac 6). These macrophage-like cells express high levels of FcgammaRI and FcgammaRII. They were shown to bind fluorescently labeled CRP with micromolar affinity, KD = (6.6 +/- 1.5) microM. CRP binding could be inhibited by pre-incubation with human but not mouse IgG and was thus FcgammaR-specific. Blocking of FcgammaRI by an FcgammaRI-specific antibody abolished CRP binding essentially completely, whereas application of antibodies against FcgammaRII did not have a noticeable effect. In fluorescence images of Mono Mac 6 cells, the intensity patterns of bound CRP were correlated with those of FcgammaRI, but not FcgammaRII. These results provide clear evidence of specific interactions between CRP and FcgammaR (predominantly FcgammaRI) naturally expressed on macrophage-like cells.
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Proteína C-Reactiva/metabolismo , Macrófagos/metabolismo , Receptores de IgG/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/análisis , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Inmunoglobulina G/metabolismo , Macrófagos/química , Unión Proteica/efectos de los fármacos , Receptores de IgG/análisis , Receptores de IgG/genética , TransfecciónRESUMEN
Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.