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Recent advancements in single‐molecule methods have not only made it possible to obtain precise measurements for complex biological processes but also to produce simple mathematical models for intricate biochemical mechanisms, which would otherwise be speculative. These developments have strengthened our ability to respond through mathematical modeling to concepts of protein‒protein and protein‒DNA interactions on a nanometer level and address‐related questions. In this article, we examine an intriguing biological phenomenon in which a protein and an enzyme co‐jointly encounter carcinogenic adducts during transcription. We are focusing mainly on the dysregulation of the protein involved and the possible consequences that may arise. By providing a quantum mechanical model, we have demonstrated that the presence of carcinogenic adducts in a transcriptional bubble deregulates the protein that could cause lethal mutations. Next, we present a case study to explore carcinogenesis by suggesting an alternative experimental design. Our quantum mechanical model emphasizes the use of a quantized energies approach for specific mechanisms within the living cells. Radiation‐induced carcinogenicity can be prevented if radiation interacting with tissue is not given the energies that satisfy the quantization conditions. [ABSTRACT FROM AUTHOR]

Abstract Recent advancements in single‐molecule methods have not only made it possible to obtain precise measurements for complex biological processes but also to produce simple mathematical models for intricate biochemical mechanisms, which would otherwise be speculative. These developments have strengthened our ability to respond through mathematical modeling to concepts of protein‒protein and protein‒DNA interactions on a nanometer level and address‐related questions. In this article, we examine an intriguing biological phenomenon in which a protein and an enzyme co‐jointly encounter carcinogenic adducts during transcription. We are focusing mainly on the dysregulation of the protein involved and the possible consequences that may arise. By providing a quantum mechanical model, we have demonstrated that the presence of carcinogenic adducts in a transcriptional bubble deregulates the protein that could cause lethal mutations. Next, we present a case study to explore carcinogenesis by suggesting an alternative experimental design. Our quantum mechanical model emphasizes the use of a quantized energies approach for specific mechanisms within the living cells. Radiation‐induced carcinogenicity can be prevented if radiation interacting with tissue is not given the energies that satisfy the quantization conditions.

Chromatin homeostasis mediates essential processes in eukaryotes, where histone chaperones have emerged as major regulatory factors during DNA replication, repair, and transcription. The dynamic nature of these processes, however, has severely impeded their characterization at the molecular level. Here, fluorescence optical tweezers are applied to follow histone chaperone dynamics in real time. The molecular action of SET/template-activating factor-l/β and nucleophosmin 1-representing the two most common histone chaperone folds-are examined using both nucleosomes and isolated histones. It is shown that these chaperones present binding specificity for fully dismantled nucleosomes and are able to recognize and disrupt non-native histone-DNA interactions. Furthermore, the histone eviction process and its modulation by cytochrome c are scrutinized. This approach shows that despite the different structures of these chaperones, they present conserved modes of action mediating nucleosome remodeling. [ABSTRACT FROM AUTHOR]

DNA is considered to be not only a carrier of the genetic information of life but also a highly programmable and self-assembled nanomaterial. Different DNA structures are related to their biological and chemical functions. Hence, understanding the physical and chemical properties of various DNA structures is of great importance in biology and nanochemistry. However, the bulk assay ignores the heterogeneity of DNA structures in solution. Single-molecule methods are powerful tools for observing the behavior of individual molecules and probing the high heterogeneity of free energy states. In this review, we introduce single-molecule methods, including single-molecule detection and manipulation methods, and discuss how these methods can be conducive to measuring the molecular properties of single-/double-stranded DNA (ss/dsDNA), DNA higher-order structures, and DNA nanostructures. We conclude by providing a new perspective on the combination of DNA nanotechnology and single-molecule methods to understand the biophysical properties of DNA and other bio-matter and soft matter.

Background fluorescence, especially when it exhibits undesired spatial features, is a primary factor for reduced image quality in optical microscopy. Structured background is particularly detrimental when analyzing single-molecule images for 3-dimensional localization microscopy or single-molecule tracking. Here, we introduce BGnet, a deep neural network with a U-net-type architecture, as a general method to rapidly estimate the background underlying the image of a point source with excellent accuracy, even when point-spread function (PSF) engineering is in use to create complex PSF shapes. We trained BGnet to extract the background from images of various PSFs and show that the identification is accurate for a wide range of different interfering background structures constructed from many spatial frequencies. Furthermore, we demonstrate that the obtained background-corrected PSF images, for both simulated and experimental data, lead to a substantial improvement in localization precision. Finally, we verify that structured background estimation with BGnet results in higher quality of superresolution reconstructions of biological structures.

The majority of proteins in nature are composed of multiple domains connected in a single polypeptide. How these long sequences fold into functional structures without forming toxic misfolds or aggregates is poorly understood. Their folding is inextricably linked to protein synthesis and interactions with cellular machinery, making mechanistic studies challenging. Recent progress has revealed critical features of multi-domain protein folding in isolation and in the context of translation by the ribosome. In this review, we discuss challenges and progress in understanding multi-domain protein folding, and highlight how molecular interactions shape folding and misfolding pathways. With the development of new approaches and model systems, the stage is now set for mechanistically exploring the folding of large multi-domain proteins.

This work presents the results of single-molecular studies of the effect of magnesium ions on the dynamic characteristics of transcription elongation of bacterial RNA polymerase. It has been shown that the instantaneous and average transcription rates decrease with a decrease in magnesium concentration. The observed dependence occurred due to an increase in the number of short pauses; an explanation of the mechanism of their formation was put forward. To carry out these studies, the method of acoustic force spectroscopy (AFS) was used. This technique served as a basis for the development of a single-molecule procedure for characterizing the transcription parameters. A detailed description of the method and algorithm for processing the measurement results was given.

[Display omitted] • AFM imaging and magnetic tweezers force spectroscopy probe tri-nucleosome arrays. • H3K36me3 and H4K5/8/12/16ac are more open than unmodified trinucleosomes. • Increased mononucleosome breathing can propagate to more open higher order chromatin. • Nucleosome-nucleosome interactions can overrule nucleosome breathing. • Unwrapping of the inner DNA turn is not affected by H3K36me3 H4K5/8/12/16ac. Nucleosomes are the basic compaction unit of chromatin and nucleosome structure and their higher-order assemblies regulate genome accessibility. Many post-translational modifications alter nucleosome dynamics, nucleosome-nucleosome interactions, and ultimately chromatin structure and gene expression. Here, we investigate the role of two post-translational modifications associated with actively transcribed regions, H3K36me3 and H4K5/8/12/16ac, in the contexts of tri-nucleosome arrays that provide a tractable model system for quantitative single-molecule analysis, while enabling us to probe nucleosome-nucleosome interactions. Direct visualization by AFM imaging reveals that H3K36me3 and H4K5/8/12/16ac nucleosomes adopt significantly more open and loose conformations than unmodified nucleosomes. Similarly, magnetic tweezers force spectroscopy shows a reduction in DNA outer turn wrapping and nucleosome-nucleosome interactions for the modified nucleosomes. The results suggest that for H3K36me3 the increased breathing and outer DNA turn unwrapping seen in mononucleosomes propagates to more open conformations in nucleosome arrays. In contrast, the even more open structures of H4K5/8/12/16ac nucleosome arrays do not appear to derive from the dynamics of the constituent mononucleosomes, but are driven by reduced nucleosome-nucleosome interactions, suggesting that stacking interactions can overrule DNA breathing of individual nucleosomes. We anticipate that our methodology will be broadly applicable to reveal the influence of other post-translational modifications and to observe the activity of nucleosome remodelers. [ABSTRACT FROM AUTHOR]

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force–distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell–Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme.

Flavin and redox-active disulfide domains of ferredoxin-dependent flavin thioredoxin reductase (FFTR) homodimers should pivot between flavin-oxidizing (FO) and flavin-reducing (FR) conformations during catalysis, but only FR conformations have been detected by X-ray diffraction and scattering techniques. Atomic force microscopy (AFM) is a single-molecule technique that allows the observation of individual biomolecules with sub-nm resolution in near-native conditions in real-time, providing sampling of molecular properties distributions and identification of existing subpopulations. Here, we show that AFM is suitable to evaluate FR and FO conformations. In agreement with imaging under oxidizing condition, only FR conformations are observed for Gloeobacter violaceus FFTR (GvFFTR) and isoform 2 of Clostridium acetobutylicum FFTR (CaFFTR2). Nonetheless, different relative dispositions of the redox-active disulfide and FAD-binding domains are detected for FR homodimers, indicating a dynamic disposition of disulfide domains regarding the central protein core in solution. This study also shows that AFM can detect morphological changes upon the interaction of FFTRs with their protein partners. In conclusion, this study paves way for using AFM to provide complementary insight into the FFTR catalytic cycle at pseudo-physiological conditions. However, future approaches for imaging of FO conformations will require technical developments with the capability of maintaining the FAD-reduced state within the protein during AFM scanning.

Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle.

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract: Monosaccharides, such as d‐glucose and d‐fructose, exist in aqueous solution as an equilibrium mixture of cyclic isomers and can be detected with boronic acids by the reversible formation of boronate esters. The engineering of accurate, discriminating and continuous monitoring devices relies on knowledge of which cyclic isomer of a sugar binds to a boronic acid receptor. Herein, by monitoring fluctuations in ionic current, we show that an engineered α‐hemolysin (αHL) nanopore modified with a boronic acid reacts reversibly with d‐glucose as the pyranose isomer (α‐ d‐glucopyranose) and d‐fructose as either the furanose (β‐ d‐fructofuranose) or the pyranose (β‐ d‐fructopyranose). Both of these binding modes contradict current binding models. With this knowledge, we distinguished the individual sugars in a mixture of d‐maltose, d‐glucose, and d‐fructose. [ABSTRACT FROM AUTHOR]

Point Spread Function (PSF) engineering is an effective method to increase the sensitivity of single-molecule fluorescence images to specific parameters. Classical phase mask optimization approaches have enabled the creation of new PSFs that can achieve, for example, localization precision of a few nanometers axially over a capture range of several microns with bright emitters. However, for complex high-dimensional optimization problems, classical approaches are difficult to implement and can be very time-consuming for computation. The advent of deep learning methods and their application to single-molecule imaging has provided a way to solve these problems. Here, we propose to combine PSF engineering and deep learning approaches to obtain both an optimized phase mask and a neural network structure to obtain the 3D position and 3D orientation of fixed fluorescent molecules. Our approach allows us to obtain an axial localization precision around 30 nanometers, as well as an orientation precision around 5 degrees for orientations and positions over a one micron depth range for a signal-to-noise ratio consistent with what is typical in single-molecule cellular imaging experiments. • Neural nets can be used to determine 3D position and orientation for single molecules. • A new optimal phase mask, the Arrowhead mask, was extracted by machine learning. • The full performance on 3D position and orientation was verified by extensive simulation. • Direct experimental measurements of single molecules in a polymer confirm performance. [ABSTRACT FROM AUTHOR]

Binding affinity is measured by dissociation constant, Kd, which uses concentration as units. The universal concentration units facilitate direct comparison of affinities for different binding events. However, Kd is a thermodynamic parameter, which lacks kinetic information of a binding event. In addition, Kd does not reveal the mechanical property of the binding, which emerges as a critical element for many physiologically significant processes such as DNA replication, RNA transcription, and protein translation. Here we propose a new parameter, mechanical affinity, to delineate kinetic and mechanical features of a binding event. The mechanical affinity is equivalent to the work required to dissemble the chemical binding between a ligand and a receptor. During this process, it must cover dissipated heat that originates from the relative movement between a ligand and a receptor. Because dissipated heat varies with unfolding direction or rate of mechanical perturbation, the mechanical affinity is a function of these two variables. Screening of chemicals using rupture force of a ligand-receptor complex or mechanical affinity is discussed at the end of this review. The interrogation on the mechanical interaction between a ligand and a receptor provides a new perspective not available in conventional thermodynamic evaluation of binding processes.

At the end of the 20th century, substantial changes in the paradigms of molecular physics and biology occurred. They have brought two new and entirely independent, fields of scientific research – nanotechnology and systems biology. Thanks to these disciplines, a new paradigm was born opening a new way of research in biology. It enables a holistic treatment of living organisms. As a consequence of these changes, an entirely new picture of the interface between physics and biology emerges.

This microreview summarizes a study aiming at the development of a novel family of metal complexes that binds to the phosphate esters of the DNA backbone inspired by the cytotoxicity of the anticancer drug cisplatin and by the phosphate ester cleaving reactivity of metalloenzymes and their biomimetic model complexes. A rational design is presented that is based on the requirements to establish a molecular recognition for the phosphate esters of the DNA backbone and to suppress binding to the less exposed nucleobases. Two phosphate binding sites should be preoriented and fixed by a rigid backbone to the distance of two neighboring phosphate ester in the DNA backbone of 6–7 Å. Sterical demand close to the phosphate binding sites should inhibit coordination to the nucleobases. This was molecularly translated into an unprecedented family of dinuclear complexes based on 1,8-naphthalenediol ligands with sterically demanding chelate arms in 2,7-position. The synthesis of the ligand and its first dinuclear Cu II 2 complex is described. The binding of this complex to DNA has been studied by biochemical ensemble methods and biophysical single-molecule methods. The incubation of DNA with the Cu II 2 complex results in interstrand interactions forming aggregates that prevent the DNA to enter the gel in the electrophoresis experiments. AFM experiments show an increase of the DNA diameter and local entanglements of the DNA by intrastrand interactions. The stretching of a single DNA molecule by optical tweezers exhibits distinct force peaks upon treatment with the complex necessary to break the intrastrand interactions. Torsional measurements of a single DNA molecule by magnetic tweezers showed a shortening of the effective DNA length due to the intrastrand interactions. Incubation with the Cu II 2 complex suggests inhibition of DNA synthesis in polymerase chain reaction experiments and strong cytotoxicity to human HeLa cancer cells, both at lower concentration than with cisplatin. A coherent model is provided that explains all experimental observations by the intended binding of the Cu II 2 complex to two neighboring phosphates of the DNA backbone and the formation of intra- or interstrand interactions by π–π interactions of the outward oriented and freely exposed naphthalene rings. [ABSTRACT FROM AUTHOR]

Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel. [ABSTRACT FROM AUTHOR]

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein-induced topological domains in DNA, quantitative and time-resolved approaches are required. Here, we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real time and at the single-molecule level. Our approach is based on quantifying the extension fluctuations-in addition to the mean extension-of supercoiled DNA in magnetic tweezers (MT). Using a combination of high-speed MT experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime, the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how the transient (partial) dissociation of DNA-bridging proteins results in the dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our results to further the understanding and optimization of magnetic tweezer measurements and to enable quantification of the dynamics and reaction pathways of DNA processing enzymes in the context of physiologically relevant forces and supercoiling densities., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

Abstract: We present a single-molecule method for measuring the torque exerted by braided DNA molecules undergoing spontaneous unbraiding while attached to a paramagnetic dumbbell in the absence of external manipulation. A magnetic tweezers setup is employed to braid pairs of lambda DNA molecules covalently bound to a surface. Upon removing the magnetic field, the braided DNA molecules undergo spontaneous unbraiding, efficiently transforming the stored elastic energy into enough mechanical energy to rotate the tethered dumbbells for periods as long as 30min. Using hydrodynamic equations we estimate the torque exerted on the dumbbells by the DNA braids, yielding values ranging from 47 to 166pNnm. [Copyright &y& Elsevier]

It has been over one-and-a-half decades since methods of single-molecule detection and manipulation were first introduced in biochemical research. Since then, the application of these methods to an expanding variety of problems has grown at a vertiginous pace. While initially many of these experiments led more to confirmatory results than to new discoveries, today single-molecule methods are often the methods of choice to establish new mechanism-based results in biochemical research. Throughout this process, improvements in the sensitivity, versatility, and both spatial and temporal resolution of these techniques has occurred hand in hand with their applications. We discuss here some of the advantages of single-molecule methods over their bulk counterparts and argue that these advantages should help establish them as essential tools in the technical arsenal of the modern biochemist. [ABSTRACT FROM AUTHOR]

Recent progress in proteornics suggests that the cell can be conceived as a large network of highly refined, nanomachine-like protein complexes. This working hypothesis calls for new methods capable of analyzing individual protein complexes in living cells and tissues at high speed. Here, we examine whether single-molecule fluorescence (SMF) analysis can satisfy that demand. First, recent technical progress in the visualization, localization, tracking, conformational analysis, and true resolution of individual protein complexes is highlighted. Second, results obtained by the SMF analysis of protein complexes are reviewed, focusing on the nuclear pore complex as an instructive example. We conclude that SMF methods provide powerful, indispensable tools for the structural and functional characterization of protein complexes. However, the transition from in vitro systems to living cells is in the initial stages. We discuss how current limitations in the nanoscopic analysis of living cells and tissues can be overcome to create a new paradigm, nanoscopic biomedicine. [ABSTRACT FROM AUTHOR]

Artificial nanopores have recently emerged as versatile tools for analyzing and sorting single molecules at high speed. However, the biological cell has already developed a large set of sophisticated protein nanopores that are able to selectively translocate all types of molecules through membranes. Therefore, hybrid devices combining artificial solid-state with biomimetic protein nanopores appear to us as a particularly promising approach to the creation of powerful diagnostic, preparative and therapeutic devices. Here, we discuss a technique, optical single-transporter recording (OSTR), in which arrays of artificial micropores and nanopores are employed to analyze protein nanopores of cellular membranes. After briefly summarizing some salient features of OSTR, the technique is compared with the electrical patch clamp method and the first results of our efforts to amalgamate optical and electrical recording are described. Finally, prospects for combining OSTR with 4Pi microscopy, single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy are discussed. [ABSTRACT FROM AUTHOR]

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force–distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell–Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme. [ABSTRACT FROM AUTHOR]

Flavin and redox-active disulfide domains of ferredoxin-dependent flavin thioredoxin reductase (FFTR) homodimers should pivot between flavin-oxidizing (FO) and flavin-reducing (FR) conformations during catalysis, but only FR conformations have been detected by X-ray diffraction and scattering techniques. Atomic force microscopy (AFM) is a single-molecule technique that allows the observation of individual biomolecules with sub-nm resolution in near-native conditions in real-time, providing sampling of molecular properties distributions and identification of existing subpopulations. Here, we show that AFM is suitable to evaluate FR and FO conformations. In agreement with imaging under oxidizing condition, only FR conformations are observed for Gloeobacter violaceus FFTR (GvFFTR) and isoform 2 of Clostridium acetobutylicum FFTR (CaFFTR2). Nonetheless, different relative dispositions of the redox-active disulfide and FAD-binding domains are detected for FR homodimers, indicating a dynamic disposition of disulfide domains regarding the central protein core in solution. This study also shows that AFM can detect morphological changes upon the interaction of FFTRs with their protein partners. In conclusion, this study paves way for using AFM to provide complementary insight into the FFTR catalytic cycle at pseudo-physiological conditions. However, future approaches for imaging of FO conformations will require technical developments with the capability of maintaining the FAD-reduced state within the protein during AFM scanning. [ABSTRACT FROM AUTHOR]

Brassinosteroids, the steroid hormones of plants, control physiological and developmental processes through its signaling pathway. The major brassinosteroid signaling network components, from the receptor to transcription factors, have been identified in the past two decades. The development of biotechnologies has driven the identification of novel brassinosteroid signaling components, even revealing several crosstalks between brassinosteroid and other plant signaling pathways. Herein, we would like to summarize the identification and improvement of several representative brassinosteroid signaling components through the development of new technologies, including brassinosteroid-insensitive 1 (BRI1), BRI1-associated kinase 1 (BAK1), BR-insensitive 2 (BIN2), BRI1 kinase inhibitor 1 (BKI1), BRI1-suppressor 1 (BSU1), BR signaling kinases (BSKs), BRI1 ethyl methanesulfonate suppressor 1 (BES1), and brassinazole resistant 1 (BZR1). Furthermore, improvement of BR signaling knowledge, such as the function of BKI1, BES1 and its homologous through clustered regularly interspaced short palindromic repeats (CRISPR), the regulation of BIN2 through single-molecule methods, and the new in vivo interactors of BIN2 identified by proximity labeling are described. Among these technologies, recent advanced methods proximity labeling and single-molecule methods will be reviewed in detail to provide insights to brassinosteroid and other phytohormone signaling pathway studies. [ABSTRACT FROM AUTHOR]

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of 'bridge over troubled ends'., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The genome-wide occurrence of G-quadruplexes and their demonstrated biological activities call for detailed understanding on the stability and transition kinetics of the structures. Although the core structural element in a G-quadruplex is simple and requires only four tandem repeats of Guanine rich sequences, there is rather rich conformational diversity in this structure. Corresponding to this structural diversity, it displays involved transition kinetics within individual G-quadruplexes and complicated interconversion among different G-quadruplex species. Due to the inherently high signal-to-noise ratio in the measurement, single-molecule tools offer a unique capability to investigate the thermodynamic, kinetic, and mechanical properties of G-quadruplexes with dynamic conformations. In this chapter, we describe different single molecule methods such as atomic-force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), optical, magnetic, and magneto-optical tweezers to investigate G-quadruplex structures as well as their interactions with small-molecule ligands.

Fluorescent nanosensors are powerful tools for basic research and bioanalytical applications. Individual nanosensors are able to detect single molecules, while ensembles of nanosensors can be used to measure the bulk concentration of an analyte. Collective imaging of multiple nanosensors could provide both spatial and temporal chemical information from the nano- to the microscale. This type of chemical imaging with nanosensors would be very attractive to study processes such as chemical signaling between cells (e.g., neurons). So far, it is not understood what processes are resolvable (concentration, time, space) and how optimal sensors should be designed. Here, we develop a theoretical framework to simulate the fluorescence image of arrays of nanosensors in response to a concentration gradient. For that purpose, binding and unbinding of the analyte is simulated for each single nanosensor by using a Monte Carlo simulation and varying rate constants (k on , k off ). Multiple nanosensors are arranged on a surface and exposed to a concentration pattern c A (x,y,t) of an analyte. We account for the resolution limit of light microscopy (Abbe limit) and the acquisition speed and resolution of optical setups and determine the resulting response images ΔI(x,y,t). Consequently, we introduce terms for the spatial and temporal resolution and simulate phase diagrams for different rate constants that allow us to predict how a sensor should be designed to provide a desired spatial and temporal resolution. Our results show, for example, that imaging of neurotransmitter release requires rate constants of k on = 10 6 M -1 s -1 and k off = 10 2 s -1 in many scenarios, which corresponds to high dissociation constants of K d > 100 μM. This work predicts if a given fluorescent nanosensor array (rate constants, size, shape, geometry, density) is able to resolve fast concentration changes such as neurotransmitter release from cells. Additionally, we provide rational design principles to engineer nanosensors for chemical imaging.

Total internal reflection fluorescence (TIRF) microscopy is a powerful technique for interrogating protein dynamics in the membranes of living single cells. Receptor-ligand interactions are of particular interest for improving our understanding of cell signaling networks in a variety of applications. Here, we describe methods for fluorescently labeling individual receptors and their ligands, conducting single-molecule TIRF microscopy of receptors and ligands in single, living cells, and importantly, performing image analysis on the resulting time sequence of images to extract quantitative dynamics. While we use Toll-like receptor 4 and its ligand lipopolysaccharide as a specific example, the methods are general and readily extendable to other receptor-ligand systems of importance in cellular biology.