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1.
Single-molecule force spectroscopy with optical tweezers has emerged as a powerful tool for dissecting protein folding. The requirement to stably attach “molecular handles” to specific points in the protein of interest by preparative biochemical techniques is a limiting factor in applying this methodology, especially for large or unstable proteins that are difficult to produce and isolate. Here, we present a streamlined approach for creating stable and specific attachments using autocatalytic covalent tethering. The high specificity of coupling allowed us to tether ribosome-nascent chain complexes, demonstrating its suitability for investigating complex macromolecular assemblies. We combined this approach with cell-free protein synthesis, providing a facile means of preparing samples for single-molecule force spectroscopy. The workflow eliminates the need for biochemical protein purification during sample preparation for single-molecule measurements, making structurally unstable proteins amenable to investigation by this powerful single-molecule technique. We demonstrate the capabilities of this approach by carrying out pulling experiments with an unstructured domain of elongation factor G that had previously been refractory to analysis. Our approach expands the pool of proteins amenable to folding studies, which should help to reduce existing biases in the currently available set of protein folding models. 相似文献
2.
A practical guide to single-molecule FRET 总被引:1,自引:0,他引:1
Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software. 相似文献
3.
Magnetic tweezers (MT) are a powerful tool for the study of DNA-enzyme interactions. Both the magnet-based manipulation and the camera-based detection used in MT are well suited for multiplexed measurements. Here, we systematically address challenges related to scaling of multiplexed magnetic tweezers (MMT) towards high levels of parallelization where large numbers of molecules (say 103) are addressed in the same amount of time required by a single-molecule measurement. We apply offline analysis of recorded images and show that this approach provides a scalable solution for parallel tracking of the xyz-positions of many beads simultaneously. We employ a large field-of-view imaging system to address many DNA-bead tethers in parallel. We model the 3D magnetic field generated by the magnets and derive the magnetic force experienced by DNA-bead tethers across the large field of view from first principles. We furthermore experimentally demonstrate that a DNA-bead tether subject to a rotating magnetic field describes a bicircular, Limaçon rotation pattern and that an analysis of this pattern simultaneously yields information about the force angle and the position of attachment of the DNA on the bead. Finally, we apply MMT in the high-throughput investigation of the distribution of the induced magnetic moment, the position of attachment of DNA on the beads, and DNA flexibility. The methods described herein pave the way to kilo-molecule level magnetic tweezers experiments. 相似文献
4.
Maarten M. van Oene Seungkyu Ha Tessa Jager Mina Lee Francesco Pedaci Jan Lipfert Nynke H. Dekker 《Biophysical journal》2018,114(8):1970-1979
Single-molecule manipulation techniques have provided unprecedented insights into the structure, function, interactions, and mechanical properties of biological macromolecules. Recently, the single-molecule toolbox has been expanded by techniques that enable measurements of rotation and torque, such as the optical torque wrench (OTW) and several different implementations of magnetic (torque) tweezers. Although systematic analyses of the position and force precision of single-molecule techniques have attracted considerable attention, their angle and torque precision have been treated in much less detail. Here, we propose Allan deviation as a tool to systematically quantitate angle and torque precision in single-molecule measurements. We apply the Allan variance method to experimental data from our implementations of (electro)magnetic torque tweezers and an OTW and find that both approaches can achieve a torque precision better than 1 pN · nm. The OTW, capable of measuring torque on (sub)millisecond timescales, provides the best torque precision for measurement times10 s, after which drift becomes a limiting factor. For longer measurement times, magnetic torque tweezers with their superior stability provide the best torque precision. Use of the Allan deviation enables critical assessments of the torque precision as a function of measurement time across different measurement modalities and provides a tool to optimize measurement protocols for a given instrument and application. 相似文献
5.
We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes. 相似文献
6.
Jorge Alegre-Cebollada Raul Perez-Jimenez Pallav Kosuri Julio M. Fernandez 《The Journal of biological chemistry》2010,285(25):18961-18966
Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed. 相似文献
7.
Jibin Abraham Punnoose Andrew Hayden Lifeng Zhou Ken Halvorsen 《Biophysical journal》2020,119(11):2231
The ability to apply controlled forces to individual molecules has been revolutionary in shaping our understanding of biophysics in areas as diverse as dynamic bond strength, biological motor operation, and DNA replication. However, the methodology to perform single-molecule experiments remains relatively inaccessible because of cost and complexity. In 2010, we introduced the centrifuge force microscope (CFM) as a platform for accessible and high-throughput single-molecule experimentation. The CFM consists of a rotating microscope with which prescribed centrifugal forces can be applied to microsphere-tethered biomolecules. In this work, we develop and demonstrate a next-generation Wi-Fi CFM that offers unprecedented ease of use and flexibility in design. The modular CFM unit fits within a standard benchtop centrifuge and connects by Wi-Fi to an external computer for live control and streaming at near gigabit speeds. The use of commercial wireless hardware allows for flexibility in programming and provides a streamlined upgrade path as Wi-Fi technology advances. To facilitate ease of use, detailed build and setup instructions, as well as LabVIEW-based control software and MATLAB-based analysis software, are provided. We demonstrate the instrument’s performance by analysis of force-dependent dissociation of short DNA duplexes of 7, 8, and 9 bp. We showcase the sensitivity of the approach by resolving distinct dissociation kinetic rates for a 7 bp duplex in which one G-C basepair is mutated to an A-T basepair. 相似文献
8.
The need for low-cost DNA sequence detection in clinical applications is driving development of new technologies. We demonstrate a method for detection of mutations in a DNA sequence purely by electronic means, and without need for fluorescent labeling. Our method uses an array of nanopores to perform synchronized single-molecule force spectroscopy measurements over many molecules in parallel, yielding detailed information on the kinetics of hundreds of molecule dissociations in a single measurement. 相似文献
9.
We report the use of an atomic force microscopy (AFM)-based force spectroscopy technique to identify, at the single-molecule level, the components of mixtures of polysaccharides. Previously, we showed that the elasticity of certain types of polysaccharides is governed by force-induced conformational transitions of the pyranose ring. These transitions produce atomic fingerprints in the force-extension spectrum that are characteristic of the ground-energy conformation of the pyranose ring and the type of glycosidic linkages. Using this approach we find that commercially available agarose and lambda-carrageenan contain molecules that, when stretched in an atomic force microscope, produce a force spectrum characteristic of alpha-(1-->4) d-glucans. We have identified these molecules as amylopectin or floridean starch, a storage polysaccharide in algae. Our methodology can identify individual polysaccharide molecules in solution, which is not possible by any other spectroscopic technique, and therefore is an important addition to the arsenal of analytical techniques used in carbohydrate research. 相似文献
10.
A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy 总被引:4,自引:0,他引:4
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In recent years, the atomic force microscope (AFM) has contributed much to our understanding of the molecular forces involved in various high-affinity receptor-ligand systems. However, a universal anchor system for such measurements is still required. This would open up new possibilities for the study of biological recognition processes and for the establishment of high-throughput screening applications. One such candidate is the N-nitrilo-triacetic acid (NTA)/His-tag system, which is widely used in molecular biology to isolate and purify histidine-tagged fusion proteins. Here the histidine tag acts as a high-affinity recognition site for the NTA chelator. Accordingly, we have investigated the possibility of using this approach in single-molecule force measurements. Using a histidine-peptide as a model system, we have determined the binding force for various metal ions. At a loading rate of 0.5 microm/s, the determined forces varied from 22 +/- 4 to 58 +/- 5 pN. Most importantly, no interaction was detected for Ca(2+) and Mg(2+) up to concentrations of 10 mM. Furthermore, EDTA and a metal ion reloading step demonstrated the reversibility of the approach. Here the molecular interactions were turned off (EDTA) and on (metal reloading) in a switch-like fashion. Our results show that the NTA/His-tag system will expand the "molecular toolboxes" with which receptor-ligand systems can be investigated at the single-molecule level. 相似文献
11.
Single-molecule force spectroscopy makes it possible to measure the mechanical strength of single noncovalent receptor-ligand-type bonds. A major challenge in this technique is to ensure that measurements reflect bonds between single biomolecules because the molecules cannot be directly observed. This perspective evaluates different methodologies for identifying and reducing the contribution of multiple molecule interactions to single-molecule measurements to help the reader design experiments or assess publications in the single-molecule force spectroscopy field. We apply our analysis to the large body of literature that purports to measure the strength of single bonds between biotin and streptavidin as a demonstration that measurements are only reproducible when the most reliable methods for ensuring single molecules are used. 相似文献
12.
Robert Crawford Joseph P. TorellaLouise Aigrain Anne PlochowietzKristofer Gryte Stephan UphoffAchillefs N. Kapanidis 《Biophysical journal》2013
Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence. 相似文献
13.
The ability to measure the properties of proteins at the single-molecule level offers an unparalleled glimpse into biological systems at the molecular scale. The interpretation of single-molecule time series has often been rooted in statistical mechanics and the theory of Markov processes. While existing analysis methods have been useful, they are not without significant limitations including problems of model selection and parameter nonidentifiability. To address these challenges, we introduce the use of nonparametric Bayesian inference for the analysis of single-molecule time series. These methods provide a flexible way to extract structure from data instead of assuming models beforehand. We demonstrate these methods with applications to several diverse settings in single-molecule biophysics. This approach provides a well-constrained and rigorously grounded method for determining the number of biophysical states underlying single-molecule data. 相似文献
14.
《Biophysical journal》2021,120(22):5018-5031
Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays while being applicable to native protein complexes isolated from single cells in vivo. 相似文献
15.
Cyclic interactions between myosin II motor domains and actin filaments that are powered by turnover of ATP underlie muscle contraction and have key roles in motility of nonmuscle cells. The elastic characteristics of actin-myosin cross-bridges are central in the force-generating process, and disturbances in these properties may lead to disease. Although the prevailing paradigm is that the cross-bridge elasticity is linear (Hookean), recent single-molecule studies suggest otherwise. Despite convincing evidence for substantial nonlinearity of the cross-bridge elasticity in the single-molecule work, this finding has had limited influence on muscle physiology and physiology of other ordered cellular actin-myosin ensembles. Here, we use a biophysical modeling approach to close the gap between single molecules and physiology. The model is used for analysis of available experimental results in the light of possible nonlinearity of the cross-bridge elasticity. We consider results obtained both under rigor conditions (in the absence of ATP) and during active muscle contraction. Our results suggest that a wide range of experimental findings from mechanical experiments on muscle cells are consistent with nonlinear actin-myosin elasticity similar to that previously found in single molecules. Indeed, the introduction of nonlinear cross-bridge elasticity into the model improves the reproduction of key experimental results and eliminates the need for force dependence of the ATP-induced detachment rate, consistent with observations in other single-molecule studies. The findings have significant implications for the understanding of key features of actin-myosin-based production of force and motion in living cells, particularly in muscle, and for the interpretation of experimental results that rely on stiffness measurements on cells or myofibrils. 相似文献
16.
Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinson’s disease, induced by misfolding of α-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of α-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized α-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected α-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded α-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, α-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of α-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of α-synuclein. 相似文献
17.
Robert Crawford Joseph?P. Torella Louise Aigrain Anne Plochowietz Kristofer Gryte Stephan Uphoff Achillefs?N. Kapanidis 《Biophysical journal》2013,105(11):2439-2450
Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence. 相似文献
18.
19.
Statistical mechanics and molecular dynamics simulations proposed that the folding of proteins can follow multiple parallel pathways on a rugged energy landscape from unfolded state en route to their folded native states. Kinetic partitioning mechanism is one of the possible mechanisms underlying such complex folding dynamics. Here, we use single-molecule atomic force microscopy technique to directly probe the multiplicity of the folding pathways of the third fibronectin type III domain from the extracellular matrix protein tenascin-C (TNfn3). By stretching individual (TNfn3)8 molecules, we forced TNfn3 domains to undergo mechanical unfolding and refolding cycles, allowing us to directly observe the folding pathways of TNfn3. We found that, after being mechanically unraveled and then relaxed to zero force, TNfn3 follows multiple parallel pathways to fold into their native states. The majority of TNfn3 fold into the native state in a simple two-state fashion, while a small percentage of TNfn3 were found to be trapped into kinetically stable folding intermediate states with well-defined three-dimensional structures. Furthermore, the folding of TNfn3 was also influenced by its neighboring TNfn3 domains. Complex misfolded states of TNfn3 were observed, possibly due to the formation of domain-swapped dimeric structures. Our studies revealed the ruggedness of the folding energy landscape of TNfn3 and provided direct experimental evidence that the folding dynamics of TNfn3 are governed by the kinetic partitioning mechanism. Our results demonstrated the unique capability of single-molecule AFM to probe the folding dynamics of proteins at the single-molecule level. 相似文献
20.
Multi-step fibrinogen binding to the integrin (alpha)IIb(beta)3 detected using force spectroscopy 总被引:1,自引:0,他引:1
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The regulated ability of integrin alphaIIbbeta3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. We have developed a model system based on laser tweezers, enabling us to measure specific rupture forces needed to separate single receptor-ligand complexes. First of all, we performed a thorough and statistically representative analysis of nonspecific protein-protein binding versus specific alphaIIbbeta3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified alphaIIbbeta3 and fibrinogen, covalently attached to underlying surfaces, ranged from approximately 20 to 150 pN. This distribution could be fit with a sum of an exponential curve for weak to moderate (20-60 pN) forces, and a Gaussian curve for strong (>60 pN) rupture forces that peaked at 80-90 pN. The interactions corresponding to these rupture force regimes differed in their susceptibility to alphaIIbbeta3 antagonists or Mn2+, an alphaIIbbeta3 activator. Varying the surface density of fibrinogen changed the total binding probability linearly >3.5-fold but did not affect the shape of the rupture force distribution, indicating that the measurements represent single-molecule binding. The yield strength of alphaIIbbeta3-fibrinogen interactions was independent of the loading rate (160-16,000 pN/s), whereas their binding probability markedly correlated with the duration of contact. The aggregate of data provides evidence for complex multi-step binding/unbinding pathways of alphaIIbbeta3 and fibrinogen revealed at the single-molecule level. 相似文献