RESUMEN
Fluctuations in mechanical force vectors within living cells can substantially influence the behavior and functions of proteins. Tau protein can spontaneously be raptured and entangled in refolding under picoNewton compressive forces that are biologically available in a living cell: a hidden aggregation pathway due to stress and crowding. Our findings were achieved through a customized modification of atomic force microscopy (AFM) for single-molecule manipulation. This previously hidden phenomenon of proteins rupturing collectively while subsequently and spontaneously refolding into a complex entangled conformation, distinct from the Tau protein's folded or unfolded states, could potentially explain the early-event initiation of the aggregation of the Tau protein seen in various neurodegenerative diseases. This article introduces our recent discovery of the missing Tau protein property that is of significant relevance to the Tau protein and neurodegenerative disease research and medical treatment, aiming to stimulate the collective observation and a new perspective on the Tau aggregation mechanism and disease mechanism studies.
Asunto(s)
Enfermedades Neurodegenerativas , Proteínas tau , Humanos , Proteínas tau/metabolismo , Agregado de Proteínas , ProteínasRESUMEN
Conformational dynamics play a crucial role in protein functions. A molecular-level understanding of the conformational transition dynamics of proteins is fundamental for studying protein functions. Here, we report a study of real-time conformational dynamic interaction between calcium-activated calmodulin (CaM) and C28W peptide using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy and imaging. Plasma membrane Ca-ATPase protein interacts with CaM by its peptide segment that contains 28 amino acids (C28W). The interaction between CaM and the Ca-ATPase is essential for cell signaling. However, details about its dynamic interaction are still not clear. In our current study, we used Cyanine3 labeled CaM (N-domain) and Dylight 649 labeled C28W peptide (N-domain) to study the conformational dynamics during their interaction. In this study, the FRET can be measured when the CaM-C28W complex is formed and only be observed when such a complex is formed. By using single-molecule FRET efficiency trajectory and unique statistical approaches, we were able to observe multiple binding steps with detailed dynamic features of loosely bound and tightly bound state fluctuations. The C-domain of CaM tends to bind with C28W first with a higher affinity, followed by the binding of the CaM N-domain. Due to the comparatively high flexibility and low affinity of the N-domain and the presence of multiple anchor hydrophobic residues on the peptide, the N-domain binding may switch between selective and non-selective binding states, while the C-domain remains strongly bound with C28W. The results provide a mechanistic understanding of the CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics.
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Calmodulina , Transferencia Resonante de Energía de Fluorescencia , Sitios de Unión , Calcio/metabolismo , Calmodulina/química , Unión Proteica , Conformación Proteica , Análisis EspectralRESUMEN
Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.
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Calmodulina , Óxido Nítrico Sintasa de Tipo III , Calmodulina/metabolismo , Transporte de Electrón , Hemo/metabolismo , Óxido Nítrico , Óxido Nítrico Sintasa , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismoRESUMEN
Mechanical force plays a critical role in the relationship between protein structure and function. Force manipulation by Atomic Force Microscope can be significant and trigger chemical and biological activities of proteins. Previously we have reported that Apo-CaM undergoes through a spontaneous tertiary structural rupture under a piconewton compressive force. Here we have observed that the ruptured Apo-CaM molecules can be available to bind with C28W peptide, a typical protein signalling activity that only a Ca2+-activated CaM has. This behaviour is both unexpected and profound, as CaM in its Ca2+-non-activated form has a closed structure which does not presumably allow the molecule to bind to target peptides. In this experiment, we demonstrate that both chemical activation and force activation can play a vital role in biology, such as the cell-signalling protein dynamics and function.
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Calmodulina/química , Proteínas/química , Transducción de Señal , Fenómenos Biomecánicos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Calmodulin (CaM) is found to have the capability to bind multiple targets. Investigations on the association mechanism of CaM to its targets are crucial for understanding protein-protein binding and recognition. Here, we developed a structure-based model to explore the binding process between CaM and skMLCK binding peptide. We found the cooperation between nonnative electrostatic interaction and nonnative hydrophobic interaction plays an important role in nonspecific recognition between CaM and its target. We also found that the conserved hydrophobic anchors of skMLCK and binding patches of CaM are crucial for the transition from high affinity to high specificity. Furthermore, this association process involves simultaneously both local conformational change of CaM and global conformational changes of the skMLCK binding peptide. We found a landscape with a mixture of the atypical "induced fit," the atypical "conformational selection," and "simultaneously binding-folding," depending on the synchronization of folding and binding. Finally, we extend our discussions on multispecific binding between CaM and its targets. These association characteristics proposed for CaM and skMLCK can provide insights into multispecific binding of CaM.
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Calmodulina/metabolismo , Calmodulina/fisiología , Quinasa de Cadena Ligera de Miosina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Calcio/metabolismo , Simulación por Computador , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica/fisiología , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas/fisiologíaRESUMEN
Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme-substrate interaction, enzyme-substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.
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Dominio Catalítico , Colorantes Fluorescentes/química , Peroxidasa de Rábano Silvestre/química , Catálisis , Transferencia Resonante de Energía de Fluorescencia , Magnetismo , Microscopía Confocal , Fotones , Conformación Proteica , Ingeniería de Proteínas , Espectrofotometría , Estrés MecánicoRESUMEN
Interrogating the protein structure-function inter-relationship under a piconewton force manipulation has been highly promising and informative. Although protein conformational changes under pulling force manipulations have been extensively studied, protein conformational changes under a compressive force have not been explored in detail. Using our home-modified sensitive and high signal-to-noise atomic force microscopy (AFM) approach, we have applied a piconewton compressive force, manipulating a Calmodulin (CaM) molecule to characterize two different forms of CaM, the Ca2+-ligated activated form and the Ca2+ free non-activated form (apo-CaM). We observed sudden and spontaneous structural rupture of apo-CaM under compressive force applied by an AFM tip, though no such events were recorded in the case of Ca2+-ligated activated CaM form. The sudden spontaneous structural rupture under a piconewton force compression has never been reported before, which presents an unexplored function that is likely important for protein-protein interactions and cell signaling functions.
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Señalización del Calcio , Calcio/química , Calmodulina/química , Fuerza Compresiva , Calcio/metabolismo , Calmodulina/metabolismo , Humanos , Microscopía de Fuerza Atómica , Conformación ProteicaRESUMEN
In this paper, we present three ratiometric near-infrared fluorescent probes (A-C) for accurate, ratiometric detection of intracellular pH changes in live cells. Probe A consists of a tetraphenylethene (TPE) donor and near-infrared hemicyanine acceptor in a through-bond energy transfer (TBET) strategy, while probes B and C are composed of TPE and hemicyanine moieties through single and double sp2 carbon-carbon bond connections in a π-conjugation modulation strategy. The specific targeting of the probes to lysosomes in live cells was achieved by introducing morpholine residues to the hemicyanine moieties to form closed spirolactam ring structures. Probe A shows aggregation-induced emission (AIE) property at neutral or basic pH, while probes B and C lack AIE properties. At basic or neutral pH, the probes only show fluorescence of TPE moieties with closed spirolactam forms of hemicyanine moieties, and effectively avoid blind fluorescence imaging spots, an issue which typical intensity-based pH fluorescent probes encounter. Three probes show ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with TPE fluorescence decreases and hemicyanine fluorescence increases, because acidic pH makes the spirolactam rings open to enhance π-conjugation of hemicyanine moieties. However, probe A shows much more sensitive ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with remarkable ratio increase of TPE fluorescence to hemicyanine fluorescence up to 238-fold than probes B and C because of its high efficiency of energy transfer from TPE donor to the hemicyanine acceptor in the TBET strategy. The probe offers dual Stokes shifts with a large pseudo-Stokes shift of 361 nm and well-defined dual emissions, and allows for colocalization of the imaging readouts of visible and near-infrared fluorescence channels to achieve more precisely double-checked ratiometric fluorescence imaging. These platforms could be employed to develop a variety of novel ratiometric fluorescent probes for accurate detection of different analytes in applications of chemical and biological sensing, imaging, and diagnostics by introducing appropriate sensing ligands to hemicyanine moieties to form on-off spirolactam switches.
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Carbocianinas/química , Colorantes Fluorescentes/química , Carbocianinas/síntesis química , Citoplasma/química , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Imagen Óptica , Espectrometría de FluorescenciaRESUMEN
The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording. Identifying the compositions of NMDA receptors, their spatial organization and distributions over live cell membranes, we observe that NMDA receptors are organized inhomogeneously: nearly half of the receptor proteins are individually dispersed; whereas others exist in heterogeneous clusters of around 50 nm in size as well as co-localized within the diffraction limited imaging area. We demonstrate that inhomogeneous interactions and partitions of the NMDA receptors can be a cause of the heterogeneous gating mechanism of NMDA receptors in living cells. Furthermore, comparing the imaging results with the ion-channel electric current recording, we propose that the clustered NMDA receptors may be responsible for the variation in the current amplitude observed in the on-off two-state ion-channel electric signal trajectories. Our findings shed new light on the fundamental structure-function mechanism of NMDA receptors and present a conceptual advancement of the ion-channel mechanism in living cells.
Asunto(s)
Receptores de N-Metil-D-Aspartato/metabolismo , Imagen Individual de Molécula/métodos , Membrana Celular , Electricidad , Células HEK293 , Humanos , Activación del Canal Iónico , Imagen Óptica/métodos , Técnicas de Placa-ClampRESUMEN
Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure-function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2-amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme-substrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions.
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Peroxidasa de Rábano Silvestre/química , Microscopía Fluorescente/métodos , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.
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Calmodulina/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Óxido Nítrico Sintasa de Tipo I/química , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Carbocianinas/metabolismo , Bovinos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrofotometría Ultravioleta , Factores de TiempoRESUMEN
Interfacial charge transfer dynamics in dye-sensitized NiO nanoparticles are being investigated for photocathodes in p-type dye-sensitized solar cells. In the photoreaction, after fast electron transfer from NiO to a molecule, the recombination of the hole in the nanoparticles with the electron in a reduced molecule plays an important role in the charge separation process and solar energy harvesting. Nevertheless, knowledge of the interfacial charge recombination (CR) rate and its mechanism is still limited due to the complex photoinduced electron and hole dynamics and lack of characterization of the inhomogeneity of the dynamics. Here, we report our work on probing interfacial charge recombination dynamics in Zn(ii)-5,10,15,20-tetra(3-carboxyphenyl)porphyrin (m-ZnTCPP) dye-sensitized NiO nanoparticles by correlating single-molecule fluorescence blinking dynamics with charge transfer dynamics using single-molecule photon-stamping spectroscopy. The correlated analyses of single-molecule fluorescence intensity, lifetime, and blinking reveal the intrinsic distribution and temporal fluctuation of interfacial charge transfer reactivity, which are closely related to site-specific molecular interactions and dynamics.
RESUMEN
N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method. Here we report the development of a new correlated technical approach, single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain. Experimentally determined kinetics of ligand binding with receptor is further verified by computational modeling. Single-channel patch-clamp and four-channel fluorescence measurement are recorded simultaneously to get correlation among electrical on and off states, optically determined conformational open and closed states by FRET, and binding-unbinding states of the glycine ligand by anisotropy measurement at the ligand binding domain of GluN1 subunit. This method has the ability to detect the intermediate states in addition to electrical on and off states. Based on our experimental results, we have proposed that NMDA receptor gating goes through at least one electrically intermediate off state, a desensitized state, when ligands remain bound at the ligand binding domain with the conformation similar to the fully open state.
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Transferencia Resonante de Energía de Fluorescencia , Glicina/metabolismo , Glicina/farmacología , Microscopía , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Anisotropía , Supervivencia Celular , Células HEK293 , Humanos , Cinética , Ligandos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Receptores de N-Metil-D-Aspartato/químicaRESUMEN
Fundamental understanding of the energetic coupling properties of a molecule-semiconductor interface is of great importance. The changes in molecular conformations and vibrational modes can have significant impact on the interfacial charge transfer reactions. Here, we have probed the change in the interface properties of alizarin-TiO2 system as a result of the externally applied electric field using single-hot spot microscopic surface-enhanced Raman spectroscopy (SMSERS) and provided a theoretical understanding of our experimental results by density functional theory (DFT) calculations. The perturbation, caused by the external potential, has been observed as a shift and splitting of the 648 cm(-1) peak, typical indicator of the strong coupling between alizarin and TiO2, at SMSERS. On the basis of our experimental results and DFT calculations, we suggest that electric field has significant effects on vibrational coupling at the molecule-TiO2 interface. The presence of perturbed alizarin-TiO2 coupling under interfacial electric potential may lead to changes in the interfacial electron transfer dynamics. Additionally, heterogeneously distributed dye molecules at the interface on nanometer length scale and different chromophore-semiconductor binding interactions under charge accumulation associated interfacial electric field changes create intrinsically inhomogeneous interfacial ET dynamics associated with both static and dynamic disorders.
RESUMEN
Enzymatic reactions and related protein conformational dynamics are complex and inhomogeneous, playing crucial roles in biological functions. The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. It is extremely difficult to characterize and analyze such complex dynamics in an ensemble-averaged measurement, especially when the enzymes are associated with multiple-step, multiple-conformation complex chemical interactions and transformations. Beyond the conventional ensemble-averaged studies, real-time single-molecule approaches have been demonstrated to be powerful in dissecting the complex enzymatic reaction dynamics and related conformational dynamics. Single-molecule enzymology has come a long way since the early demonstrations of the single-molecule spectroscopy studies of enzymatic dynamics about two decades ago. The rapid development of this fundamental protein dynamics field is hand-in-hand with the new development of single-molecule imaging and spectroscopic technology and methodology, theoretical model analyses, and correlations with biological preparation and characterization of the enzyme protein systems. The complex enzymatic reactions can now be studied one molecule at a time under physiological conditions. Most exciting developments include active manipulation of enzymatic conformational changes and energy landscape to regulate and manipulate the enzymatic reactivity and associated conformational dynamics, and the new advancements have established a new stage for studying complex protein dynamics beyond by simply observing but by actively manipulating and observing the enzymatic dynamics at the single-molecule sensitivity temporally and spatially.
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Biocatálisis , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía de Fuerza Atómica/métodos , Microscopía Fluorescente/métodos , Proteínas/química , Animales , Diseño de Equipo , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Humanos , Microscopía de Fuerza Atómica/instrumentación , Microscopía Fluorescente/instrumentación , Simulación de Dinámica Molecular , Imagen Óptica/instrumentación , Imagen Óptica/métodos , Conformación Proteica , Proteínas/metabolismoRESUMEN
Conformational dynamics plays a critical role in the activation, deactivation, and open-close activities of ion channels in living cells. Such conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy, to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging with single-channel electric current recording. Our approach is particularly capable of resolving ion channel conformational change rate process when the channel is at its electrically off states and before the ion channel is activated, the so-called "silent time" when the electric current signals are at zero or background. We have probed NMDA (N-methyl-D-aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. On the basis of our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics.
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Receptores de N-Metil-D-Aspartato/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Microscopía Fluorescente , Técnicas de Placa-Clamp , Conformación Proteica , Receptores de N-Metil-D-Aspartato/análisisRESUMEN
Enzyme-substrate interaction plays a critical role in enzymatic reactions, forming the active enzyme-substrate complex, the transition state ready to react. Studying the enzyme-substrate interaction will help in the ultimate molecular-level characterization of the enzymatic transition state that defines the reaction pathway, energetics, and the dynamics. In our initial effort to experimentally investigate the enzyme-substrate interactions and the related conformational fluctuations, we have developed a new approach to manipulate the enzymatic conformation and enzyme-substrate interaction at a single-molecule level by using a combined magnetic tweezers and simultaneous fluorescence resonance energy transfer (FRET) spectroscopic microscopy. By a repetitive pulling-releasing manipulation of a Cy3-Cy5 dye labeled 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) molecule under the conditions with and without enzymatic substrates, we have probed and analyzed the enzymatic conformational dynamics. Our results indicate that the enzyme conformational flexibility can be regulated by enzyme-substrate interactions: (1) enzyme at its conformation-perturbed state has less flexibility when binding substrates, and (2) substrate binding to enzyme significantly changes the enzyme conformational flexibility, an experimental evidence of so called entropy trapping in the enzyme-substrate reactive transition state. Furthermore, our results provide a significant experimental analysis of the folding-binding enzyme-substrate interactions, a dynamic nature of the enzymatic active transition state formation process.
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Difosfotransferasas/química , Difosfotransferasas/ultraestructura , Transferencia Resonante de Energía de Fluorescencia/métodos , Magnetismo/métodos , Micromanipulación/métodos , Microscopía/métodos , Espectrometría de Fluorescencia/métodos , Activación Enzimática , Técnicas de Sonda Molecular , Conformación Proteica , Especificidad por SustratoRESUMEN
Making and holding an artificial lipid bilayer horizontally in an aqueous solution within the microscopic working distance of ~100 µm are essential for simultaneous single molecule imaging and single ion-channel electrical current recording. However, preparation of such a lipid bilayer without a solid support is technically challenging. In a typical supported lipid bilayer, the asymmetric local environments and the strong perturbation of the underneath solid or dense surface can diverge the normal behavior of membrane proteins and lipids. On the other hand, the suspended lipid bilayers can provide a native local environment for the membrane proteins and lipids by having fluids on both sides. In this technical report, we present a simple and novel methodology for making a suspended lipid bilayer that can be used for recording the single-molecule diffusion and single ion-channel electrical measurements of ion-channel proteins. Our approach has a higher validity for studying the molecular diffusions and conformational fluctuations of membrane proteins without having perturbations from supporting layers. We demonstrate the feasibility of such an approach on simultaneous single-molecule fluorescence imaging and electric current measurements of ion channel proteins.
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Colicinas/química , Canales Iónicos/química , Membrana Dobles de Lípidos/química , Colicinas/metabolismo , Difusión , Impedancia Eléctrica , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Modelos Biológicos , Fenómenos ÓpticosRESUMEN
We investigate the single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic, highly soluble Cr(VI) can be efficiently reduced to less toxic, nonsoluble Cr(2)O(3) nanoparticles by MR-1. Cr(2)O(3) is observed to emerge as nanoparticles adsorbed on the cell surface and its chemical nature is identified by EDX imaging and Raman spectroscopy. Co-localization of Cr(2)O(3) and cytochromes by EDX imaging and Raman spectroscopy suggests a terminal reductase role for MR-1 surface-exposed cytochromes MtrC and OmcA. Our experiments revealed that the cooperation of surface proteins OmcA and MtrC makes the reduction reaction most efficient, and the sequence of the reducing reactivity of MR-1 is wild type > single mutant ΔmtrC or mutant ΔomcA > double mutant (ΔomcA-ΔmtrC). Moreover, our results also suggest that direct microbial Cr(VI) reduction and Fe(II) (hematite)-mediated Cr(VI) reduction mechanisms may coexist in the reduction processes.
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Compuestos de Cromo/química , Shewanella/metabolismo , Compuestos de Cromo/metabolismo , Grupo Citocromo c/metabolismo , Microscopía Electrónica de Rastreo , Nanopartículas/química , Oxidación-Reducción , Shewanella/química , Espectrometría por Rayos X , Espectrometría Raman , Propiedades de SuperficieRESUMEN
Light harvesting by LH1 and LH2 antenna proteins in the photosynthetic membranes of purple bacteria has been extensively studied in recent years for the fundamental understanding of the energy transfer dynamics and mechanism. Here we report the inhomogeneous structural organization of the LH2 complexes in photosynthetic membranes, giving evidence for the existence of energetically coupled linear LH2 aggregates in the native photosynthetic membranes of purple bacteria. Focusing on systematic model analyses, we combined AFM imaging and spectroscopic analysis with energetic coupling model analysis to characterize the inhomogeneous linear aggregation of LH2. Our AFM imaging results reveal that the LH2 complexes form linear aggregates with the monomer number varying from one to eight and each monomer tilted along the aggregated structure in photosynthetic membranes. The spectroscopic results support the attribution of aggregated LH2 complexes in the photosynthetic membranes, and the model calculation values for the absorption, emission and lifetime are consistent with the experimentally determined spectroscopic values, further proving a molecular-level understanding of the energetic coupling and energy transfer among the LH2 complexes in the photosynthetic membranes.