RESUMEN
Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.
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Transferencia Resonante de Energía de Fluorescencia , Proteínas , Transferencia Resonante de Energía de Fluorescencia/métodos , Reproducibilidad de los Resultados , Proteínas/química , Conformación Molecular , LaboratoriosRESUMEN
Previous works have reported significant effects of macromolecular crowding on the structure and behavior of biomolecules. The crowded intracellular environment, in contrast to in vitro buffer solutions, likely imparts similar effects on biomolecules. The enzyme serving as the gatekeeper for the genome, RNA polymerase (RNAP), is among the most regulated enzymes. Although it was previously demonstrated that macromolecular crowding affects association of RNAP to DNA, not much is known about how crowding acts on late initiation and promoter clearance steps, which are considered to be the rate-determining steps for many promoters. Here, we demonstrate that macromolecular crowding enhances the rate of late initiation and promoter clearance using in vitro quenching-based single-molecule kinetics assays. Moreover, the enhancement's dependence on crowder size notably deviates from predictions by the scaled-particle theory, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions could be affected by crowded conditions in the cellular milieu.
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Proteínas de Unión al ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Transcripción Genética , Citoplasma/enzimología , Citoplasma/genética , Proteínas de Unión al ADN/química , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/enzimología , Escherichia coli/genética , Genoma Bacteriano/genética , Cinética , Sustancias Macromoleculares/química , Regiones Promotoras Genéticas , TermodinámicaRESUMEN
Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used o increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.
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ADN/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Imagen Individual de Molécula/métodos , Simulación por Computador , ARN Polimerasas Dirigidas por ADN/química , Difusión , Ensayos Analíticos de Alto Rendimiento/métodos , Cinética , Láseres de Semiconductores , Microfluídica/métodos , Conformación Molecular , Fotones , Iniciación de la Transcripción GenéticaRESUMEN
Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.
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Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , ARN Polimerasa II/genética , ARN Mensajero/genética , Iniciación de la Transcripción Genética , Secuencia de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Conformación de Ácido Nucleico , ARN Polimerasa II/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
Single-molecule fluorescence detection (SMFD) experiments are useful in distinguishing sub-populations of molecular species when measuring heterogeneous samples. One experimental platform for SMFD is based on a confocal microscope, where molecules randomly traverse an effective detection volume. The non-uniformity of the excitation profile and the random nature of Brownian motion, produce fluctuating fluorescence signals. For these signals to be distinguished from the background, burst analysis is frequently used. Yet, the relation between the results of burst analyses and the underlying information of the diffusing molecules is still obscure and requires systematic assessment. In this work we performed three-dimensional Brownian motion simulations of SMFD, and tested the positions at which molecules emitted photons that passed the burst analysis criteria for different values of burst analysis parameters. The results of this work verify which of the burst analysis parameters and experimental conditions influence both the position of molecules in space when fluorescence is detected and taken into account, and whether these bursts of photons arise purely from single molecules, or not entirely. Finally, we show, as an example, the effect of bursts that are not purely from a single molecule on the accuracy in single-molecule Förster resonance energy transfer measurements.
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Microscopía Confocal , Microscopía Fluorescente , Imagen Individual de Molécula , Transferencia Resonante de Energía de Fluorescencia , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Modelos Teóricos , FotonesRESUMEN
Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few "structural snapshots" along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique's temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.
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ARN Polimerasas Dirigidas por ADN/química , Modelos Biológicos , Regiones Promotoras Genéticas , ARN Polimerasas Dirigidas por ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Conformación Molecular , Regiones Promotoras Genéticas/genéticaRESUMEN
The initiation of transcription in Escherichia coli (E. coli) is facilitated by promoter specificity factors, also known as σ factors, which may bind a promoter only as part of a complex with RNA polymerase (RNAP). By performing in vitro cross-linking mass spectrometry (CL-MS) of apo-σ70, we reveal structural features suggesting a compact conformation compared to the known RNAP-bound extended conformation. Then, we validate the existence of the compact conformation using in vivo CL-MS by identifying cross-links similar to those found in vitro, which deviate from the extended conformation only during the stationary phase of bacterial growth. Conclusively, we provide information in support of a compact conformation of apo-σ70 that exists in live cells, which might represent a transcriptionally inactive form that can be activated upon binding to RNAP.
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Phytoplankton are a major source of primary productivity. Their photosynthetic fluorescence are unique measures of their type, physiological state, and response to environmental conditions. Changes in phytoplankton photophysiology are commonly monitored by bulk fluorescence spectroscopy, where gradual changes are reported in response to different perturbations, such as light intensity changes. What is the meaning of such trends in bulk parameters if their values report ensemble averages of multiple unsynchronized cells? To answer this, we developed an experimental scheme that enables tracking fluorescence intensities, brightnesses, and their ratios, as well as mean photon nanotimes equivalent to mean fluorescence lifetimes, one cell at a time. We monitored three different phytoplankton species during diurnal cycles and in response to an abrupt increase in light intensity. Our results show that we can define specific subpopulations of cells by their fluorescence parameters for each of the phytoplankton species, and in response to varying light conditions. Importantly, we identify the cells undergo well-defined transitions between these subpopulations. The approach shown in this work will be useful in the exact characterization of phytoplankton cell states and parameter signatures in response to different changes these cells experience in marine environments, which will be applicable for monitoring marine-related environmental effects.
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Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and opensource Brick-MIC platform will be a valuable tool for many laboratories worldwide.
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Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we report the development of a flow virometer for the specific and rapid detection of single nanoparticles based on confocal microscopy. The combination of laminar flow and multiple dyes enable the detection of correlated fluorescence signals, providing information on nanoparticle volumes and specific chemical composition properties, such as viral envelope proteins. We evaluated and validated the assay using fluorescent beads and viruses, including SARS-CoV-2. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting clinically-relevant virus loads. Based on our results, we envision the use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup.
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Small amyloid-ß (Aß) oligomers have much higher membrane affinity compared to the monomers, but the structural origin of this functional change is not understood. We show that as monomers assemble into small n-mers (n < 10), Aß acquires a tertiary fold that is consistent with the mature fibrils. This is an early and defining transition for the aggregating peptide, and possibly underpins its altered bioactivity.
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Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/química , Fluoresceína/química , Transferencia Resonante de Energía de Fluorescencia , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosfatidilcolinas/química , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1α (HP1α) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1α condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.
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Núcleo Celular , Células Madre Embrionarias , Animales , Ratones , Núcleo Celular/metabolismo , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Heterocromatina/metabolismo , Homólogo de la Proteína Chromobox 5RESUMEN
Parkinson disease is associated with the aggregation of the protein α-synuclein. While α-synuclein can exist in multiple oligomeric states, the dimer has been a subject of extensive debates. Here, using an array of biophysical approaches, we demonstrate that α-synuclein in vitro exhibits primarily a monomer-dimer equilibrium in nanomolar concentrations and up to a few micromolars. We then use spatial information from hetero-isotopic cross-linking mass spectrometry experiments as restrains in discrete molecular dynamics simulations to obtain the ensemble structure of dimeric species. Out of eight structural sub-populations of dimers, we identify one that is compact, stable, abundant, and exhibits partially exposed ß-sheet structures. This compact dimer is the only one where the hydroxyls of tyrosine 39 are in proximity that may promote dityrosine covalent linkage upon hydroxyl radicalization, which is implicated in α-synuclein amyloid fibrils. We propose that this α-synuclein dimer features etiological relevance to Parkinson disease.
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Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Conformación Molecular , Amiloide/químicaRESUMEN
PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.
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ADN , Proteínas , ADN/química , Proteínas/química , Transferencia Resonante de Energía de FluorescenciaRESUMEN
PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.
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The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66-86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Aminoácidos/metabolismo , Animales , Difusión , Humanos , Ratones , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Dispersión del Ángulo Pequeño , Factores de Tiempo , Difracción de Rayos XRESUMEN
Single-molecule spectroscopy has revolutionized molecular biophysics and provided means to probe how structural moieties within biomolecules spatially reorganize at different timescales. There are several single-molecule methodologies that probe local structural dynamics in the vicinity of a single dye-labeled residue, which rely on fluorescence lifetimes as readout. Nevertheless, an analytical framework to quantify dynamics in such single-molecule single dye fluorescence bursts, at timescales of microseconds to milliseconds, has not yet been demonstrated. Here, we suggest an analytical framework for identifying and quantifying within-burst lifetime-based dynamics, such as conformational dynamics recorded in single-molecule photo-isomerization-related fluorescence enhancement. After testing the capabilities of the analysis on simulations, we proceed to exhibit within-burst millisecond local structural dynamics in the unbound α-synuclein monomer. The analytical framework provided in this work paves the way for extracting a full picture of the energy landscape for the coordinate probed by fluorescence lifetime-based single-molecule measurements.
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Single molecule Förster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacromolecules. Photon-by-photon hidden Markov modeling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may cause artifacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species, all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin.
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Transferencia Resonante de Energía de Fluorescencia , Fotones , Conformación MolecularRESUMEN
Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the α-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled α-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled α-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while α-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.
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Conformación Molecular , Transferencia Resonante de Energía de Fluorescencia , Proteínas Intrínsecamente Desordenadas , Sustancias Macromoleculares , SinucleínasRESUMEN
α-Synuclein plays an important role in synaptic functions by interacting with synaptic vesicle membrane, while its oligomers and fibrils are associated with several neurodegenerative diseases. The specific monomer structures that promote its membrane binding and self-association remain elusive due to its transient nature as an intrinsically disordered protein. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.