Single-molecule biophysics

The concept of optical antennas in physical optics is still evolving. Like the antennas used in the radio frequency (RF) regime, the aspiration of optical antennas is to localize the free propagating radiation energy, and vice versa. For this purpose, optical antennas utilize the distinctive properties of metal nanostructures, which are strong plasmonic coupling elements at the optical regime. The concept of optical antennas is being advanced technologically and they are projected to be substitute devices for detection in the millimeter, infrared, and visible regimes. At present, their potential benefits in light detection, which include polarization dependency, tunability, and quick response times have been successfully demonstrated. Optical antennas also can be seen as directionally responsive elements for point detectors. This review provides an overview of the historical background of the topic, along with the basic concepts and parameters of optical antennas. One of the major parts of this review covers the use of optical antennas in biosensing, presenting biosensing applications with a broad description using different types of data. We have also mentioned the basic challenges in the path of the universal use of optical biosensors, where we have also discussed some legal matters.

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on single-stranded DNA (ssDNA) in an ATP dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (E. coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. The ribosome is a large cellular complex that synthesizes proteins. During assembly of the small (30S) subunit of the E. coli ribosome, 20 ribosomal proteins associate with the 16S rRNA in a defined hierarchy 1-3 that arises from protein-induced changes in the structure of the rRNA 4. Despite progress in visualizing ribosome assembly intermediates 5 , the physical Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

1.2 Importance of enzyme dynamicsinterconversion between states 2 1.3 Techniques that measure travel time between enzymatic states 4 1.4 SWCNT-FET devices can study microsecond conformational changes 9 1.5 Bioconjugation strategies for device fabrication 10 1.6 References 13 CHAPTER 2: Monitoring the Single-Molecule Dynamics of PKA by 17-54 Functionalization of Single-Walled Carbon Nanotube Circuits 2.1 Abstract 17 2.2 Introduction 18 2.3 Engineering a single cysteine variant of PKA 22 2.4 Experimental setup 28 2.5 Control electronic measurements of PKA 29 2.6 Electronic measurements of PKA binary and ternary complexes 31 2.7 Electrically-driven deposition of peptides onto SWCNTs 39 iv 2.8 PKA and caveolin-1 binding studies 2.9 Conclusions 2.10 Materials and methods 2.11 References CHAPTER 3: Improving the Cysteine-Maleimide Bioconjugation 55-81 for SWCNT-FETs

Author(s): Iftikhar, Mariam | Advisor(s): Weiss, Gregory A | Abstract: Molecular motions of proteins and their flexibility induce conformational states required for enzyme catalysis, signal transduction, and protein-protein interactions. However, the mechanisms for protein transitions between conformational states are often poorly understood, especially in the millisecond to microsecond range where conventional optical techniques and computational modeling are most limited. To investigate the microsecond dynamics of enzymes, single-walled carbon nanotube – field-effect transistors (SWCNT-FETs) were used as single-molecule biosensors. The SWCNT-FETs have sufficient sensitivity and bandwidth to monitor the conformational motions and processivity of an individual cAMP-dependent protein kinase A (PKA) molecule. Protein attachment is accomplished by functionalizing a SWCNT-FET device with a single protein and measuring the conductance versus time through the device as it is submerged in ...

Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. Thes...

RecA is a central enzyme of homologous recombination in bacteria, which plays a major role in DNA repair, natural transformation and SOS-response activation. RecA forms nucleoprotein filaments on single-stranded DNA with a highly conserved architecture that is also shared by eukaryotic recombinases. One of the key features of these filaments is the ability to switch between stretched and compressed conformations in response to ATP binding and hydrolysis. However, the functional role of such conformational changes is not fully understood. Structural data revealed that in the absence of ATP RecA binds DNA with the stoichiometry of 5 nucleotides per one monomer, while in the presence of ATP the binding stoichiometry is 3:1. Such differences suggest incompatibility of the active and inactive conformations, yet dynamic single-molecule studies demonstrated that ATP and apo conformations can be directly interconvertible. In the present work we use a single-molecule approach to address the features of inactive RecA nucleoprotein filaments formed de novo in the absence of nucleotide cofactors. We show that compressed RecA-DNA filaments can exist with both 5:1 and 3:1 binding stoichiometry which is determined by conditions of the filament assembly. However, only a 3:1 stoichiometry allows direct interconvertibility with the active ATP-bound conformation.

The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through assembly of long helical filaments around singlestranded DNA in the presence of ATP. Large-scale conformational changes induced by ATP hydrolysis result in transitions between stretched and compressed forms of the filament. Here, using a single-molecule approach, we show that compressed RecA nucleoprotein filaments can exist in two distinct interconvertible states depending on the presence of ADP in the monomermonomer interface. Binding of ADP promotes cooperative conformational transitions and directly affects mechanical properties of the filament. Our findings reveal that RecA nucleoprotein filaments are able to continuously cycle between three mechanically distinct states that might have important implications for RecA-mediated processes of HR.

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on ssDNA in an ATP-dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (Escherichia coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity. Editor's evaluation This paper is of interest to readers in the fields of DNA repair, DNA-protein interactions, and those employing single-molecule techniques. Using single-molecule methods, the authors visualized how RecX, a negative regulator of homologous recombination in bacteria, interferes with the active species in recombination, the RecA nucleoprotein filament. They showed that RecX binds to the RecA filament in its post-ATP hydrolysis state, promotes RecA dissociation from ssDNA, and causes a reversible conformational change of the filament. The latter mode of RecX action is novel and of particular interest. The authors present an interesting model of the RecX-RecA-ATP-ssDNA system.

Experiments to measure a single molecule/particle, i.e., an individual molecule/particle, at room temperature or under physiological conditions without immobilization—for example, on a surface or without significant hydrodynamic flow—have so far failed. This failure has given impetus to the underlying theory of Brownian molecular motion towards its stochastics due to diffusion. Quantifying the thermodynamic jitter of molecules/particles inspires many and forms the theoretical basis of single-molecule/single-particle biophysics and biochemistry. For the first time, our simulation results for a live cell (cytoplasm) show that the tracks of individual single molecules are localized in Brownian motion, while there is fanning out in fractal diffusion (anomalous diffusion).

In microscopy and analytical instrumentation, a researcher or clinician may need to excite several fluorophores in a sample in order to generate a useful fluorescence map of the cell or tissue of interest. This typically would include a nuclear marker such as DAPI, a green-emitting fluorophore such as FITC or GFP, and a red-emitting fluorophore such as mCherry, TRITC, or Texas Red. These red-emitting dyes can be efficiently excited using an arc lamp with a strong peak in the 550 nm region and another at 580 nm. When technology in microscope light sources originally moved to light-emitting diodes (LEDs) in the early 2000s, fluorescence work in the green gap (540 to 590 nm) excitation range was a challenge with no solution. This article explains how technology has adapted to provide LEDs that match the excitation of fluorophores typically used in multiplex fluorescence imaging.

Real-time three-dimensional single-particle tracking (RT-3D-SPT) allows continuous detection of individual freely diffusing objects with high spatiotemporal precision by applying closed-loop active feedback in an optical microscope. However, the current tracking speed in RT-3D-SPT is primarily limited by the response time of control actuators, impeding long-term observation of fast diffusive objects such as single molecules. Here, we present an RT-3D-SPT system with improved tracking performance by replacing the XY piezoelectric stage with a galvo scanning mirror with an approximately five-time faster response rate (~5 kHz). Based on the previously developed 3D single-molecule active real-time tracking (3D-SMART), this new implementation with a fast-responding galvo mirror eliminates the mechanical movement of the sample and allows more rapid response to particle motion. The improved tracking performance of the galvo mirror-based implementation is verified through simulation and pro...

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of “locking on” to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART was applied to actively track single Atto 647N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking was further applied to single proteins and nucleic acids, directly measuring the diffusion coefficient of various lengths (99 to 1385 bp) of single DNA molecules at speeds up to 10 μm2/s. In addition, 3D-SMART was able to quantify to occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.

The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 a...

We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long term accessibility of data from single-molecule fluorescence experiments based on photon-counting detectors such as single-photon avalanche diode (SPAD), photomultiplier tube (PMT) or arrays of such detectors. The format is based on HDF5, a widely used platform-and language-independent hierarchical file format for which user-friendly viewers are available. Photon-HDF5 can store raw photon data (timestamp, channel number, etc…) from any acquisition hardware, but also setup and sample description, information on provenance, authorship and other metadata, and is flexible enough to include any kind of custom data. The format specifications are hosted on a public website, which is open to contributions by the biophysics community. As an initial resource, the website provides code examples to read Photon-HDF5 files in several programming languages and a reference Python library (phconvert), to create new Photon-HDF5 files and convert several existing file formats into Photon-HDF5. To encourage adoption by the academic and commercial communities, all software is released under the MIT open source license. .

Microfluidic devices (MDs) have emerged as novel analytical tools in many areas of science and industry. Their inherent qualities such as low power requirements, low sample consumption, rapid and parallel analysis, and automation provides unique opportunities to create novel and more powerful devices with a myriad of applications. Although MDs based on PDMS have been widely used, there is still a need to develop other mechanisms for the manipulation and injection of small volumes of sample in PDMS structrues. Herein, we describe a simple, external inline magnetic valve for use in MDs constructed of PDMS and employing small magnets.

In single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence, a phenomenon known as blinking. Intermittent emissions create multiple localizations belonging to the same molecule, resulting in blinking-artifacts within SMLM images. These artifacts are often interpreted as true biological assemblies, confounding quantitative analyses and interpretations. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequenc...

We investigated the effect of the point substitutions in the N-terminal domain of the yeast prion protein Sup35 (Sup35NMp) on the structure of its amyloid fibrils. As the objects of the study, proteins with mutations that have different influence on the [PSI + ] prion propagation, but do not prevent the aggregation of Sup35NMp in vitro were chosen. The use of the wide range of physico-chemical methods allowed us to show significant differences in the structure of these aggregates, their physical size, clumping tendency. Also we demonstrated that the fluorescent probe thioflavin T can be successfully used for investigation of subtle changes in the structural organization of fibrils formed from various Sup35NMp. The obtained results and our theoretical predictions allowed us to conclude that some of selected amino acid substitutions delimit the region of the protein that forms the core of amyloid fibrils, and change the fibrils structure. The relationship of structural features of Sup35NMp amyloid aggregates, prepared in vitro, with the stability of the [PSI + ] prion in vivo allowed us to suggest that oligopeptide repeats (R) of the amyloidogenic N-terminal domain of Sup35NMp from R0 to R2 play a key role in protein aggregation. Their arrangement rather than just presence is critical for propagation of the strong [PSI + ] prion variants. The results confirm the suitability of the proposed combination of theoretical and empirical approaches for identifying changes in the amyloid fibrils structure, which, in turn, can significantly affect both the functional stability of amyloid fibrils and their pathogenicity.

Prion and some other incurable human neurodegenerative diseases are associated with misfolding of specific proteins, followed by the formation of amyloids. Despite the widespread usage of the transmission electron and of the atomic force microscopy for studing such amyloids, many related methodological issues still have not been studied until now. Here, we consider one of the first amyloids found in Saccharomyces cerevisiae yeast, i.e. Sup35NMp, to study the adsorption of monomeric protein and its fibrils on the surface of mica, silica, gold and on formvar film. Comparison of linear characteristics of these units calculated by processing of images obtained by the atomic force, transmission and scanning electron microscopy was carried out. The minimal number of measurements of fibril diameters to obtain the values in a given confidence interval were determined. We investigated the film formed by monomeric protein on mica surface, which veiled some morphology features of fibrils. Besides, we revealed that parts of the Sup35NMp excluded from the fibril core can form a wide "coat". The length of the protein forming the core of the fibrils was estimated.

The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents. Recently isolated antibodies recognizing highly conserved epitopes on the HA protein that are critical for the protein's fusogenic capacity are able to neutralize infection from a broad range of influenza subtypes. Binding of these antibodies to a virus prior to inducing fusion with a target membrane resulted in inhibition of the fusion process, directly confirming one mechanism of infection neutralization. Fluorescently labeling the antibodies allowed for functional stoichiometric measurements that indicate a virion can be rendered non-fusogenic without the need for antibodies to bind and inactivate every HA present on the viral surface. A molecular model of fusion inhibition is proposed wherein coordination between neighboring HA is disrupted that leads to neutralization of the entire particle.

The diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description associating the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here, we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature, and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to sub-diffusive behaviour. These experimental observations and the transition from Brownian to sub-diffusive motion can be recapitulated in a minimal phenomenological model.Methods Cell culture. HeLa and NIH 3T3 cells were cultured in 1.0 g • l-1 glucose, phenol-red free DMEM (11054020, Gibco) supplemented with 10% (v/v) FBS (10270106, Gibco), 1× GlutaMAX™ (35050061, Gibco), and 100 U • ml-1 of penicillin/streptomycin (15140122, Gibco). Primary normal, adult Human Dermal Fibroblast (HDFa, PCS-201-012™, ATCC ®) were cultured in 4.5 g • l-1 glucose, phenol-red free DMEM (31053028, Gibco) supplemented with 10% (v/v) FCS (CVFSVF00-01, Eurobio), 1 mM Sodium Pyruvate (11360070, Gibco), 100 U • ml-1 penicillin/streptomycin (15140122, Gibco), and 1× GlutaMAX™ (35050061, Gibco). Retinal Pigmented Epithelial (RPE-1) cells were cultivated using DME/F-12 medium (D8437, Sigma-Aldrich) supplemented with 10% (v/v) FBS (10270106, Gibco). Cells were passed every 3 days when reaching 80-90% confluency. Human Mesenchymal Stem Cells (hMSC, MSC-001F, StemCell) were cultivated with MesenCult™ proliferation kit (05411, StemCell) in flask coated with type I collagen (C8919, Sigma-Aldrich). Cells were passed every 10 days when reaching 70% confluency. All cells were maintained at 37°C in humidified, CO 2-controlled (5%) incubators. HeLa cells were purchased from ATCC. NIH 3T3 and hMSC cells were provided by Maïté Coppey-Moisan (Institut Jacques Monod) and originally obtained from ATCC and StemCell, respectively. RPE-1 cells were provided by Bruno Goud (Institut Curie) and originally obtained from ATCC. HDFa cells were provided by Sébastien Letard and Patrice Dubreuil (Centre de Recherche en Cancérologie de Marseille) and originally obtained from ATCC.

The concept of optical antennas in physical optics is still evolving. Like the antennas used in the radio frequency (RF) regime, the aspiration of optical antennas is to localize the free propagating radiation energy, and vice versa. For this purpose, optical antennas utilize the distinctive properties of metal nanostructures, which are strong plasmonic coupling elements at the optical regime. The concept of optical antennas is being advanced technologically and they are projected to be substitute devices for detection in the millimeter, infrared, and visible regimes. At present, their potential benefits in light detection, which include polarization dependency, tunability, and quick response times have been successfully demonstrated. Optical antennas also can be seen as directionally responsive elements for point detectors. This review provides an overview of the historical background of the topic, along with the basic concepts and parameters of optical antennas. One of the major parts of this review covers the use of optical antennas in biosensing, presenting biosensing applications with a broad description using different types of data. We have also mentioned the basic challenges in the path of the universal use of optical biosensors, where we have also discussed some legal matters.

electronic devices like field-effect transistors (FETs) have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes (SWNTs) have the requisite sensitivity to detect single molecule events, and have sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of polymerase I. Initial successes in each case indicate the generality and attractiveness of SWNT FETs as a new tool to complement other single molecule techniques. Furthermore, our focused research on transduction mechanisms provides the design rules necessary to further generalize this SWNT FET technique. This presentation will summarize these rules, and demonstrate how the purposeful incorporation of just one amino acid is sufficient to fabricate effective, single molecule nanocircuits from a wide range of enzymes or proteins.

Highlights d RNA polymerase and other DNA translocases can push Mcm2-7 DH complexes along DNA d Mcm2-7 DH complexes remain functional after displacement along DNA d DNA replication can initiate from sites distal to ARS elements in budding yeast d Regulated transcription termination maintains intergenic replication origins

Bacillus Calmette Guerin (BCG) vaccines comprise a family of related strains. Whole genome sequencing has allowed the better characterisation of the differences between many of the BCG vaccines. As sequencing technologies improve, updating of publicly available sequence data becomes common practice. We hereby announce the draft genome of four commonly used BCG vaccines in Brazil, Argentina and Venezuela.

To better understand the folding transitions in TTR we undertook single molecule force spectroscopy measurements of wild type (WT) TTR and two mutants (Y78F and L55P). All three TTR variants exhibit different susceptibilities to form amyloid aggregates in the pH range of 3.6-7.4 at which measurements were carried out. The resulting force-extension curves were characterized by closely spaced transitions presumably arising from the successive unfolding of secondary structure elements along a single polypeptide chain. Each of these transitions corresponds therefore to an unfolding intermediate. We observed that the frequencies with which these intermediates occurred depended on the experimental conditions. In addition, mutations in the TTR sequence greatly affect the stability of these intermediates as demonstrated by differences intermediate unfolding-force in a manner that may explain their ability to form amyloid aggregates at different pHs.

Deformation of the cell surface hinders the ability of AFM to image at molecular resolution. The damping of the cantilever determines the minimum applied force. We have developed submicron cantilevers and sensitive detection systems to reduce the damping and image at highest resolution. We have also developed active scan algorithms and data augmentation methods which enable high-speed data acquisition for capturing protein dynamics in the membrane.

Deformation of the cell surface hinders the ability of AFM to image at molecular resolution. The damping of the cantilever determines the minimum applied force. We have developed submicron cantilevers and sensitive detection systems to reduce the damping and image at highest resolution. We have also developed active scan algorithms and data augmentation methods which enable high-speed data acquisition for capturing protein dynamics in the membrane.

The dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those induced by microtubule-destabilizing agents and physical barriers. Mammalian CLASPs encompass three TOG-like domains, TOG1, TOG2, and TOG3, none of which bind to free tubulin. TOG2 is essential for catastrophe suppression, whereas TOG3 mildly enhances rescues but cannot suppress catastrophes. These functions are inhibited by the C-terminal domain of CLASP2, while the TOG1 domain can release this auto-inhibition. TOG2 fused to a positively charged microtubule-binding peptide autonomously accumulates at growing but not shrinking ends, suppresses catastrophes, and stimulates rescues. CLASPs suppress catastrophes by stabilizing growing microtubule e...

The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely used in biophysical/chemical assays and drug treatments. We present single-molecule experiments and a three-state statistical mechanical model that provide a quantitative understanding of the interplay between B-DNA, overstretched DNA and intercalated DNA. The predictions of this model include a hitherto unconfirmed hyperstretched state, twice the length of B-DNA. Our force-fluorescence experiments confirm this hyperstretched state and reveal its sequence dependence. These results pin down the physical principles that govern DNA mechanics under the influence of tension and biochemical reactions. A predictive understanding of the possibilities and limitations of DNA extension can guide re...

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein’s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.

In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al. 1 and from Lerner, Chung et al. 2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1-2 nucleotide backtracking of a 7-mer transcript.

ABSTRACTAdvanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (viaPIFE) and energy transfer efficiency (viaFRET) can simultaneously report on e.g., the conformational state of dsDNA following its interaction with unlabelled proteins (BamHI, EcoRV, T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schem...

Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3–10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores.While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by so.ware implementation. In particular, despite the widespread application of smFRET, no complete so.ware ...

We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long-term accessibility of data from single-molecule fluorescence experiments based on photon-counting detectors such as single-photon avalanche diode, photomultiplier tube, or arrays of such detectors. The format is based on HDF5, a widely used platform- and language-independent hierarchical file format for which user-friendly viewers are available. Photon-HDF5 can store raw photon data (timestamp, channel number, etc.) from any acquisition hardware, but also setup and sample description, information on provenance, authorship and other metadata, and is flexible enough to include any kind of custom data. The format specifications are hosted on a public website, which is open to contributions by the biophysics community. As an initial resource, the website provides code examples to read Photon-HDF5 files in several programming languages and a reference Python library (phconvert), to create new Photon...

Helicases are molecular engines which translocate along nucleic acids (NA) to unwind double-strands or remodel NA-protein complexes. While they have an essential role in genome structure and expression, the rules dictating their processivity remain elusive. Here, we developed single-molecule methods to investigate helicase binding lifetime on DNA. We found that UPF1, a highly processive helicase central to nonsense-mediated mRNA decay (NMD), tightly holds onto NA, allowing long lasting action. Conversely, the structurally similar IGHMBP2 helicase has a short residence time. UPF1 mutants with variable grip on DNA show that grip tightness dictates helicase residence time and processivity. In addition, we discovered via functional studies that a decrease in UPF1 grip impairs NMD efficiency in vivo. Finally, we propose a three-state model with bound, sliding and unbound molecular clips, that can accurately predict the modulation of helicase processivity.

Super-resolution microscopy (SRM) is a prime tool to study chromatin organisation at near biomolecular resolution in the native cellular environment. With fluorescent labels DNA, chromatin-associated proteins and specific epigenetic states can be identified with high molecular specificity. The aim of this review is to introduce the field of diffraction-unlimited SRM to enable an informed selection of the most suitable SRM method for a specific chromatin-related research question. We will explain both diffraction-unlimited approaches (coordinate-targeted and stochastic-localisation-based) and list their characteristic spatio-temporal resolutions, live-cell compatibility, image-processing, and ability for multi-colour imaging. As the increase in resolution, compared to, e.g. confocal microscopy, leads to a central role of the sample quality, important considerations for sample preparation and concrete examples of labelling strategies applicable to chromatin research are discussed. To ...

Disulfide bonds are formed as posttranslational modifications in a third of human proteins. The biological significance of disulfide formation is further underscored by its critical importance in numerous pathological processes including bacterial infection, viral assembly and protein misfolding disease. In eukaryotic cells, protein synthesis takes place in the cytosol where thioredoxin (TRX) prevents the formation of disulfides. Oxidative folding occurs mainly in the endoplasmic reticulum (ER) and is catalyzed by the thioredoxin-like oxidase Protein Disulfide Isomerase (PDI). Previous studies have suggested the engagement of PDI with unfolded substrates during ongoing ER translocation. How

Bacillus Calmette Guerin (BCG) vaccines comprise a family of related strains. Whole genome sequencing has allowed the better characterisation of the differences between many of the BCG vaccines. As sequencing technologies improve, updating of publicly available sequence data becomes common practice. We hereby announce the draft genome of four commonly used BCG vaccines in Brazil, Argentina and Venezuela.

Single molecule localization microscopy (SMLM) allows unprecedented insight into the three-dimensional organization of proteins at the nanometer scale. The combination of minimal invasive cell imaging with high resolution positions SMLM at the forefront of scientific discovery in cancer, infectious, and degenerative diseases. By stochastic temporal and spatial separation of light emissions from fluorescent labelled proteins, SMLM is capable of nanometer scale reconstruction of cellular structures. Precise localization of proteins in 3D astigmatic SMLM is dependent on parameter sensitive preprocessing steps to select regions of interest. With SMLM acquisition highly variable over time, it is non-trivial to find an optimal static parameter configuration. The high emitter density required for reconstruction of complex protein structures can compromise accuracy and introduce artifacts. To address these problems, we introduce two modular auto-tuning pre-processing methods: adaptive signal detection and learned recurrent signal density estimation that can leverage the information stored in the sequence of frames that compose the SMLM acquisition process. We show empirically that our contributions improve accuracy, precision and recall with respect to the state of the art. Both modules auto-tune their hyper-parameters to reduce the parameter space for practitioners, improve robustness and reproducibility, and are validated on a reference in silico dataset. Adaptive signal detection and density prediction can offer a practitioner, in addition to informed localization, a tool to tune acquisition parameters ensuring improved reconstruction of the underlying protein complex. We illustrate the challenges faced by practitioners in applying SMLM algorithms on real world data markedly different from the data used in development and show how ERGO can be run on new datasets without retraining while motivating the need for robust transfer learning in SMLM.

ABSTRACTThe search for a promoter on DNA by RNA polymerase (RNAP) is an obligatory first step in transcription. The role of facilitated diffusion during promoter search has been controversial. Here, we re-assessed facilitated diffusion in promoter search by imaging motions of single molecules of Escherichia coli RNAP σ70 holoenzyme on single DNA molecules suspended between optical traps in a manner that absolutely avoided interactions with surfaces. The assay enabled us to observe unambiguous one-dimensional sliding of RNAP σ70 holoenzyme for thousands of DNA base pairs during promoter search. Analysis of binding kinetics revealed short binding events on nonspecific DNA (0.4 s), intermediate binding events on A/T-rich DNA (1.6 s), and long binding events at or near promoters (>300 s). We estimate a lower bound for the “diffusion facilitation threshold” – the RNAP concentration at which three-dimensional search and one-dimensional sliding contribute equally to promoter binding – o...