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The integrity of the protective mucus layer as a primary defense against pathogen invasion and microbial leakage into the intestinal epithelium can be compromised by the effects of antibiotics on the commensal microbiome. Changes in mucus integrity directly affect the solvent viscosity in the immediate vicinity of the mucin network, that is, the nanoviscosity, which in turn affects both biochemical reactions and selective transport. To assess mucus nanoviscosity, a reliable readout via the viscosity-dependent fluorescence lifetime of the molecular rotor dye cyanine 3 is established and nanoviscosities from porcine and murine ex vivo mucus are determined. To account for different mucin concentrations due to the removal of digestive residues during mucus collection, the power law dependence of mucin concentration on viscosity is used. The impact of antibiotics combinations (meropenem/vancomycin, gentamycin/ampicillin) on ex vivo intestinal mucus nanoviscosity is presented. The significant increase in viscosity of murine intestinal mucus after treatment suggests an effect of antibiotics on the microbiota that affects mucus integrity. This method will be a useful tool to assess how drugs, directly or indirectly, affect mucus integrity. Additionally, the method can be utilized to analyze the role of mucus nanoviscosity in health and disease, as well as in drug development.
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Phytochromes are bistable red/far-red light-responsive photoreceptor proteins found in plants, fungi, and bacteria. Light-activation of the prototypical phytochrome Cph1 from the cyanobacterium Synechocystis sp. PCC 6803 allows photoisomerization of the bilin chromophore in the photosensory module and a subsequent series of intermediate states leading from the red absorbing Pr to the far-red-absorbing Pfr state. We show here via osmotic and hydrostatic pressure-based measurements that hydration of the photoreceptor modulates the photoconversion kinetics in a controlled manner. While small osmolytes like sucrose accelerate Pfr formation, large polymer osmolytes like PEG 4000 delay the formation of Pfr. Thus, we hypothesize that an influx of mobile water into the photosensory domain is necessary for proceeding to the Pfr state. We suggest that protein hydration changes are a molecular event that occurs during photoconversion to Pfr, in addition to light activation, ultrafast electric field changes, photoisomerization, proton release and uptake, and the major conformational change leading to signal transmission, or simultaneously with one of these events. Moreover, we discuss this finding in light of the use of Cph1-PGP as a hydration sensor, e.g., for the characterization of novel hydrogel biomaterials.
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Materiales Biocompatibles , Fitocromo , Ósmosis , Transporte Biológico , ElectricidadRESUMEN
The rapid development of microscopic techniques over the past decades enables the establishment of single molecule fluorescence imaging as a powerful tool in biological and biomedical sciences. Single molecule fluorescence imaging allows to study the chemical, physicochemical, and biological properties of target molecules or particles by tracking their molecular position in the biological environment and determining their dynamic behavior. However, the precise determination of particle distribution and diffusivities is often challenging due to high molecule/particle densities, fast diffusion, and photobleaching/blinking of the fluorophore. A novel, accurate, and fast statistical analysis tool, Diffusion Analysis of NAnoscopic Ensembles (DANAE), that solves all these obstacles is introduced. DANAE requires no approximations or any a priori input regarding unknown system-inherent parameters, such as background distributions; a requirement that is vitally important when studying the behavior of molecules/particles in living cells. The superiority of DANAE with various data from simulations is demonstrated. As experimental applications of DANAE, membrane receptor diffusion in its natural membrane environment, and cargo mobility/distribution within nanostructured lipid nanoparticles are presented. Finally, the method is extended to two-color channel fluorescence microscopy.
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Nanotecnología , Imagen Individual de Molécula , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , DifusiónRESUMEN
Phytochromes are biological red/far-red light sensors found in many organisms. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY, PGP) and the C-terminal transmitter (output) module. We recently showed a direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains of the prototypical phytochrome Cph1 PGP. These results suggested that the transient phycocyanobilin (PCB) chromophore deprotonation is closely associated with a higher protein mobility both in proximal and distal protein sites, implying a causal relationship that might be important for the global large-scale protein rearrangements. Here, we investigate the prototypical biliverdin (BV)-binding phytochrome Agp1. The structural changes at various positions in Agp1 PGP were investigated as a function of pH using picosecond time-resolved fluorescence anisotropy and site-directed fluorescence labeling of cysteine variants of Agp1 PGP. We show that the direct correlation of chromophore deprotonation with pH-dependent conformational changes does not occur in Agp1. Together with the absence of long-range effects between the PHY domain and chromophore pKa, in contrast to the findings in Cph1, our results imply phytochrome species-specific correlations between transient chromophore deprotonation and intramolecular signal transduction.
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Fitocromo , Fitocromo/química , Conformación Molecular , Luz , Cisteína , Proteínas Bacterianas/metabolismoRESUMEN
Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.
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Rodopsinas Microbianas , Bases de Schiff , Animales , Hidrógeno , Enlace de Hidrógeno , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Bases de Schiff/química , Análisis EspectralRESUMEN
Modern biology investigations on phytochromes as near-infrared fluorescent pigments pave the way for the development of new biosensors, as well as for optogenetics and in vivo imaging tools. Recently, near-infrared fluorescent proteins (NIR-FPs) engineered from biliverdin-binding bacteriophytochromes and cyanobacteriochromes, and from phycocyanobilin-binding cyanobacterial phytochromes have become promising probes for fluorescence microscopy and in vivo imaging. However, current NIR-FPs typically suffer from low fluorescence quantum yields and short fluorescence lifetimes. Here, we applied the rational approach of combining mutations known to enhance fluorescence in the cyanobacterial phytochrome Cph1 to derive a series of highly fluorescent variants with fluorescence quantum yield exceeding 15%. These variants were characterised by biochemical and spectroscopic methods, including time-resolved fluorescence spectroscopy. We show that these new NIR-FPs exhibit high fluorescence quantum yields and long fluorescence lifetimes, contributing to their bright fluorescence, and provide fluorescence lifetime imaging measurements in E.coli cells.
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Fitocromo , Proteínas Bacterianas/metabolismo , Biliverdina/química , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Fitocromo/químicaRESUMEN
Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr â Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.
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Proteínas Bacterianas/metabolismo , Pigmentos Biliares/metabolismo , Fotorreceptores Microbianos/metabolismo , Fitocromo/química , Proteínas Quinasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Pigmentos Biliares/química , Histidina Quinasa/metabolismo , Luz , Conformación Molecular , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/ultraestructura , Fitocromo/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/ultraestructura , Transducción de Señal , Synechocystis/metabolismo , Tetrapirroles/metabolismoRESUMEN
Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain of mitochondria and many aerobic prokaryotes that function as a redox-coupled proton pump, catalyzes the reduction of molecular oxygen to water. As part of the respiratory chain, CcO contributes to the proton motive force driving ATP synthesis. While many aspects of the enzyme's catalytic mechanisms have been established, a clear picture of the proton exit pathway(s) remains elusive. Here, we aim to gain insight into the molecular mechanisms of CcO through the development of a new homologous mutagenesis/expression system in Paracoccus denitrificans, which allows mutagenesis of CcO subunits 1, 2, and 3. Our system provides true single thiol-reactive CcO variants in a three-subunit base variant with unique labeling sites for the covalent attachment of reporter groups sensitive to nanoenvironmental factors like protonation, polarity, and hydration. To this end, we exchanged six residues on both membrane sides of CcO for cysteines. We show redox-dependent wetting changes at the proton uptake channel and increased polarity at the proton exit side of CcO upon electronation. We suggest an electronation-dependent conformational change to play a role in proton exit from CcO.
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Complejo IV de Transporte de Electrones/química , Fluorescencia , Imagen Óptica , Protones , Adenosina Trifosfato/biosíntesis , Adenosina Trifosfato/química , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Electrones , Modelos Moleculares , Paracoccus denitrificans/enzimología , Conformación ProteicaRESUMEN
Nanoparticles hold a great promise in biomedical science. However, due to their unique physical and chemical properties they can lead to overproduction of intracellular reactive oxygen species (ROS). As an important mechanism of nanotoxicity, there is a great need for sensitive and high-throughput adaptable single-cell ROS detection methods. Here, fluorescence lifetime imaging microscopy (FLIM) is employed for single-cell ROS detection (FLIM-ROX) providing increased sensitivity and enabling high-throughput analysis in fixed and live cells. FLIM-ROX owes its sensitivity to the discrimination of autofluorescence from the unique fluorescence lifetime of the ROS reporter dye. The effect of subcytotoxic amounts of cationic gold nanoparticles in J774A.1 cells and primary human macrophages on ROS generation is investigated. FLIM-ROX measures very low ROS levels upon gold nanoparticle exposure, which is undetectable by the conventional method. It is demonstrated that cellular morphology changes, elevated senescence, and DNA damage link the resulting low-level oxidative stress to cellular adverse effects and thus nanotoxicity. Multiphoton FLIM-ROX enables the quantification of spatial ROS distribution in vivo, which is shown for skin tissue as a target for nanoparticle exposure. Thus, this innovative method allows identifying of low-level ROS in vitro and in vivo and, subsequently, promotes understanding of ROS-associated nanotoxicity.
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Nanopartículas del Metal/toxicidad , Nanopartículas/toxicidad , Imagen Óptica/métodos , Estrés Oxidativo/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , ADN/metabolismo , Oro/toxicidad , Células HeLa , Humanos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Targeted topical application promises high drug concentrations in the skin and low systemic adverse effects. To locate drugs and drug-delivery systems like nanocarriers, fluorescent dyes are commonly used as drug surrogates or nanocarrier labels in micrographs of tissue sections. Here, we investigate how labeling degree, concentration of fluorophore, and nanocarrier may affect the interpretation of these micrographs. False-negative penetration results due to inter- and intramolecular quenching effects are likely. Using tecto-dendrimers as an example, we present a detailed analysis of pitfalls in the (semi-)quantitative evaluation of skin nanocarrier penetration. Fluorescence lifetime imaging microscopy (FLIM) allows distinguishing the target fluorescence of dye-tagged nanocarriers from skin autofluorescence, providing a highly sensitive tool for clear-cut localization of the nanocarriers. Cluster-FLIM images reveal that FITC-labeled tecto-dendrimers penetrate the stratum corneum of human skin ex vivo and reconstructed human skin but do not cross the tight junction barrier.
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Nanoestructuras , Imagen Óptica/métodos , Piel/diagnóstico por imagen , Uniones Estrechas/metabolismo , Dendrímeros , Sistemas de Liberación de Medicamentos , Fluorescencia , Colorantes Fluorescentes , HumanosRESUMEN
Phytochromes are biological red/far-red light sensors found in many organisms. Photoisomerization of the linear methine-bridged tetrapyrrole triggers transient proton translocation events in the chromophore binding pocket (CBP) leading to major conformational changes of the protein matrix that are in turn associated with signaling. By combining pH-dependent resonance Raman and UV-visible absorption spectroscopy, we analyzed protonation-dependent equilibria in the CBP of Cph1 involving the proposed Pr-I and Pr-II substates that prevail below and above pH 7.5, respectively. The protonation pattern and vibrational properties of these states were further characterized by means of hybrid quantum mechanics/molecular mechanics calculations. From this combined experimental-theoretical study, we were able to identify His260 as the key residue controlling pH-dependent equilibria. This residue is not only responsible for the conformational heterogeneity of CBP in the Pr state of prokaryotic phytochromes, discussed extensively in the past, but it constitutes the sink and source of protons in the proton release/uptake mechanism involving the tetrapyrrole chromophore which finally leads to the formation of the Pfr state. Thus, this work provides valuable information that may guide further experiments toward the understanding of the specific role of protons in controlling structure and function of phytochromes in general.
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Proteínas Bacterianas/química , Cianobacterias/química , Fitocromo/química , Proteínas Quinasas/química , Protones , Sitios de Unión , Fotorreceptores Microbianos , Conformación Proteica , Teoría CuánticaRESUMEN
A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.
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Chlamydomonas reinhardtii/química , Luz , Proteínas de Plantas/química , Rodopsina/química , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Rodopsina/genética , Rodopsina/metabolismoRESUMEN
We report here on the application of laser-based single molecule total internal reflection fluorescence microscopy (TIRFM) to study the penetration of molecules through the skin. Penetration of topically applied drug molecules is often observed to be limited by the size of the respective drug. However, the molecular mechanisms which govern the penetration of molecules through the outermost layer of the skin are still largely unknown. As a model compound we have chosen a larger amphiphilic molecule (fluorescent dye ATTO-Oxa12) with a molecular weight >700 Da that was applied to excised human skin. ATTO-Oxa12 penetrated through the stratum corneum (SC) into the viable epidermis as revealed by TIRFM of cryosections. Single particle tracking of ATTO-Oxa12 within SC sheets obtained by tape stripping allowed us to gain information on the localization as well as the lateral diffusion dynamics of these molecules. ATTO-Oxa12 appeared to be highly confined in the SC lipid region between (intercellular space) or close to the envelope of the corneocytes. Three main distinct confinement sizes of 52 ± 6, 118 ± 4, and 205 ± 5 nm were determined. We conclude that for this amphiphilic model compound several pathways through the skin exist.