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Sample records for single protein molecule

  1. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    (NCS1). The NMR solution structure of NCS1, in combination with fluorescence spectroscopy and mutational analysis, suggested a novel role for the C-terminal tail in regulating conformational stability. On the single-molecule level, the C-domain folded through a partially folded intermediate state....... This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical...

  2. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    . This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical......, with transition states located almost halfway between the native and unfolded states. When pulled from the N- and C-termini, both experiments and simulations suggested that the molecule populates a transition state that resembles that observed during chemical denaturation, with respect to structure and position...

  3. Stochastic single-molecule dynamics of synaptic membrane protein domains

    Science.gov (United States)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  4. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  5. Dynamic protein assemblies in homologous recombination with single DNA molecules

    NARCIS (Netherlands)

    van der Heijden, A.H.

    2007-01-01

    What happens when your DNA breaks? This thesis describes experimental work on the single-molecule level focusing on the interaction between DNA and DNA-repair proteins, in particular bacterial RecA and human Rad51, involved in homologous recombination. Homologous recombination and its central event

  6. Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

    CERN Document Server

    Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

    2001-01-01

    Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

  7. Single-molecule mechanics of protein-labelled DNA handles

    Directory of Open Access Journals (Sweden)

    Vivek S. Jadhav

    2016-01-01

    Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular

  8. Single-molecule studies of unconventional motor protein myosin VI

    Science.gov (United States)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  9. Single Protein Molecule Mapping with Magnetic Atomic Force Microscopy

    Science.gov (United States)

    Moskalenko, Andriy V.; Yarova, Polina L.; Gordeev, Sergey N.; Smirnov, Sergey V.

    2010-01-01

    Abstract Understanding the structural organization and distribution of proteins in biological cells is of fundamental importance in biomedical research. The use of conventional fluorescent microscopy for this purpose is limited due to its relatively low spatial resolution compared to the size of a single protein molecule. Atomic force microscopy (AFM), on the other hand, allows one to achieve single-protein resolution by scanning the cell surface using a specialized ligand-coated AFM tip. However, because this method relies on short-range interactions, it is limited to the detection of binding sites that are directly accessible to the AFM tip. We developed a method based on magnetic (long-range) interactions and applied it to investigate the structural organization and distribution of endothelin receptors on the surface of smooth muscle cells. Endothelin receptors were labeled with 50-nm superparamagnetic microbeads and then imaged with magnetic AFM. Considering its high spatial resolution and ability to “see” magnetically labeled proteins at a distance of up to 150 nm, this approach may become an important tool for investigating the dynamics of individual proteins both on the cell membrane and in the submembrane space. PMID:20141762

  10. Tetramethylammonium-filled protein nanopore for single-molecule analysis.

    Science.gov (United States)

    Wang, Ying; Yao, Fujun; Kang, Xiao-feng

    2015-10-06

    Nanopore technology, as the simplest and most inexpensive single-molecule tool, is being intensively developed. In nanopore stochastic sensing, KCl and NaCl have traditionally been employed as pore-filled electrolytes for recording the change of ion conductance in nanopores triggered by analyte translocation through the pore. However, some challenges limit its further advance. Here we used tetramethylammonium (TMA) chloride, instead of KCl, as a novel analysis system for nanopores. Some unique nanopore characteristics were observed: (1) The stability of the planar lipid bilayer for embedding the protein pores was elevated at least 6 times. (2) The TMA-Cl system could effectively reduce the noise of single-channel recording. (3) It was easy to control the insertion of protein pores into the lipid bilayer, and the formed single nanopore could last for a sufficiently long time. (4) TMA-Cl could be used as a DNA speed bump in the nanopore to slow DNA translocation speed. (5) The capacity of the nanopore capture of DNA (capture rate) increased significantly and simultaneously increased the translocation time of DNA in the pore. (6) The TMA-filled nanopore could discriminate between various polynucleotides.

  11. Single-Molecule Study of Proteins by Biological Nanopore Sensors

    Science.gov (United States)

    Wu, Dongmei; Bi, Sheng; Zhang, Liyu; Yang, Jun

    2014-01-01

    Nanopore technology has been developed for detecting properties of proteins through monitoring of ionic current modulations as protein passes via a nanosize pore. As a real-time, sensitive, selective and stable technology, biological nanopores are of widespread concern. Here, we introduce the background of nanopore researches in the area of α-hemolysin (α-HL) nanopores in protein conformation detections and protein–ligand interactions. Moreover, several original biological nanopores are also introduced with various features and functions. PMID:25268917

  12. Single-Molecule Study of Proteins by Biological Nanopore Sensors

    Directory of Open Access Journals (Sweden)

    Dongmei Wu

    2014-09-01

    Full Text Available Nanopore technology has been developed for detecting properties of proteins through monitoring of ionic current modulations as protein passes via a nanosize pore. As a real-time, sensitive, selective and stable technology, biological nanopores are of widespread concern. Here, we introduce the background of nanopore researches in the area of α-hemolysin (α-HL nanopores in protein conformation detections and protein–ligand interactions. Moreover, several original biological nanopores are also introduced with various features and functions.

  13. Single-molecule protein sequencing through fingerprinting: computational assessment

    NARCIS (Netherlands)

    Yao, Y.; Docter, M.; Ginkel, van J.; Ridder, de D.; Joo, C.

    2015-01-01

    Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has

  14. Single-molecule measurements and dynamical simulations of protein molecules near silicon substrates

    International Nuclear Information System (INIS)

    Hanasaki, Itsuo; Kawano, Satoyuki; Takahashi, Hiroto; Sazaki, Gen; Nakajima, Kazuo

    2008-01-01

    Interactions between protein molecules and inorganic substrates were studied both experimentally and numerically to obtain fundamental insight into the assembly of biomacromolecules for engineering applications. We experimentally traced individual fluorescent-labelled lysozyme (F-lysozyme) molecules, diffusing in the vicinity of interfaces between a protein solution and oxidized Si(1 0 0) and glass plates. The results indicate that diffusion coefficients of F-lysozyme molecules on both substrates are more than three orders of magnitude smaller than those in a bulk solution. The molecular dynamics simulations reveal a drastically diminished diffusion coefficient of lysozyme on the substrates of pure Si(1 1 1) and oxidized Si(1 0 0) with a hydroxy-terminated surface compared with that in bulk solution due to molecular adsorption behaviour on the substrate, which is in good agreement with experimental results. Furthermore, full atomistic description of the behaviour provides detailed information of deformation due to the adsorption process. Lysozyme on pure Si(1 1 1) undergoes substantial deformation whereas that on oxidized Si(1 0 0) does not, which indicates the importance of substrate surface condition to preserve the structure, i.e. functionality of adsorbed biomolecules

  15. See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

    NARCIS (Netherlands)

    van Mameren, J.; Peterman, E.J.G.; Wuite, G.J.L.

    2008-01-01

    Direct visualization of DNA and proteins allows researchers to investigate DNA-protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of

  16. Single molecules and nanotechnology

    CERN Document Server

    Vogel, Horst

    2007-01-01

    This book focuses on recent advances in the rapidly evolving field of single molecule research. These advances are of importance for the investigation of biopolymers and cellular biochemical reactions, and are essential to the development of quantitative biology. Written by leading experts in the field, the articles cover a broad range of topics, including: quantum photonics of organic dyes and inorganic nanoparticles their use in detecting properties of single molecules the monitoring of single molecule (enzymatic) reactions single protein (un)folding in nanometer-sized confined volumes the dynamics of molecular interactions in biological cells The book is written for advanced students and scientists who wish to survey the concepts, techniques and results of single molecule research and assess them for their own scientific activities.

  17. Shedding light on protein folding, structural and functional dynamics by single molecule studies

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean...... property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out...

  18. Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

    Directory of Open Access Journals (Sweden)

    Krutika Bavishi

    2014-11-01

    Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

  19. Revealing Two-State Protein-Protein Interaction of Calmodulin by Single-Molecule Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H PETER.

    2006-08-09

    We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of 28 amino-acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM-C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (F1AsH) that enables our unambiguously probing the CaM N-terminal target-binding domain motions at a millisecond timescale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between F1AsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal slow (at sub-second time scale) binding-unbinding motions of the N-terminal domain of the CaM in CaM-C28W complexes, which is a strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

  20. Two states or not two states: Single-molecule folding studies of protein L

    Science.gov (United States)

    Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

    2018-03-01

    Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

  1. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  2. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Science.gov (United States)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  3. Single-Molecule Characterization of DNA-Protein Interactions Using Nanopore Biosensors.

    Science.gov (United States)

    Squires, A H; Gilboa, T; Torfstein, C; Varongchayakul, N; Meller, A

    2017-01-01

    Detection and characterization of nucleic acid-protein interactions, particularly those involving DNA and proteins such as transcription factors, enzymes, and DNA packaging proteins, remain significant barriers to our understanding of genetic regulation. Nanopores are an extremely sensitive and versatile sensing platform for label-free detection of single biomolecules. Analyte molecules are drawn to and through a nanoscale aperture by an electrophoretic force, which acts upon their native charge while in the sensing region of the pore. When the nanopore's diameter is only slightly larger than the biopolymer's cross section (typically a few nm); the latter must translocate through the pore in a linear fashion due to the constricted geometry in this region. These features allow nanopores to interrogate protein-nucleic acids in multiple sensing modes: first, by scanning and mapping the locations of binding sites along an analyte molecule, and second, by probing the strength of the bond between a protein and nucleic acid, using the native charge of the nucleic acid to apply an electrophoretic force to the complex while the protein is geometrically prevented from passing through the nanopore. In this chapter, we describe progress toward nanopore sensing of protein-nucleic acid complexes in the context of both mapping binding sites and performing force spectroscopy to determine the strength of interactions. We conclude by reviewing the strengths and challenges of the nanopore technique in the context of studying DNA-protein interactions. © 2017 Elsevier Inc. All rights reserved.

  4. Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

    Directory of Open Access Journals (Sweden)

    Huijun Xue

    2017-10-01

    Full Text Available Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA and origin recognition complex (ORC as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

  5. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    IAS Admin

    RESONANCE. February 2015. GENERAL ARTICLE. Single-Molecule Spectroscopy. Every Molecule is Different! Kankan Bhattacharyya. Keywords. Single-molecule ..... Resonance Energy. Transfer (FRET) is an elegant technique to measure the distance between a donor and an acceptor molecule. FRET refers to the.

  6. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    Science.gov (United States)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  7. Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

    Science.gov (United States)

    2015-01-01

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  8. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    IAS Admin

    GENERAL ARTICLE. Single-Molecule Spectroscopy. Every Molecule is Different! Kankan Bhattacharyya. Keywords. Single-molecule spectroscopy. (SMS), confocal microscopy,. FCS, sm-FRET, FLIM. 1 High-resolution spectrum re- fers to a spectrum consisting of very sharp lines. The sharp lines clearly display transitions to ...

  9. Watching single molecules dance

    Science.gov (United States)

    Mehta, Amit Dinesh

    Molecular motors convert chemical energy, from ATP hydrolysis or ion flow, into mechanical motion. A variety of increasingly precise mechanical probes have been developed to monitor and perturb these motors at the single molecule level. Several outstanding questions can be best approached at the single molecule level. These include: how far does a motor progress per energy quanta consumed? how does its reaction cycle respond to load? how many productive catalytic cycles can it undergo per diffusional encounter with its track? and what is the mechanical stiffness of a single molecule connection? A dual beam optical trap, in conjunction with in vitro ensemble motility assays, has been used to characterize two members of the myosin superfamily: muscle myosin II and chick brain myosin V. Both move the helical polymer actin, but myosin II acts in large ensembles to drive muscle contraction or cytokinesis, while myosin V acts in small numbers to transport vesicles. An optical trapping apparatus was rendered sufficiently precise to identify a myosin working stroke with 1nm or so, barring systematic errors such as those perhaps due to random protein orientations. This and other light microscopic motility assays were used to characterize myosin V: unlike myosin II this vesicle transport protein moves through many increments of travel while remaining strongly bound to a single actin filament. The step size, stall force, and travel distance of myosin V reveal a remarkably efficient motor capable of moving along a helical track for over a micrometer without significantly spiraling around it. Such properties are fully consistent with the putative role of an organelle transport motor, present in small numbers to maintain movement over long ranges relative to cellular size scales. The contrast between myosin II and myosin V resembles that between a human running on the moon and one walking on earth, where the former allows for faster motion when in larger ensembles but for less

  10. Measuring the force of single protein molecule detachment from surfaces with AFM.

    Science.gov (United States)

    Tsapikouni, Theodora S; Missirlis, Yannis F

    2010-01-01

    Atomic force microscopy (AFM) was used to measure the non-specific detachment force of single fibrinogen molecules from glass surfaces. The identification of single unbinding events was based on the characteristics of the parabolic curves, recorded during the stretching of protein molecules. Fibrinogen molecules were covalently bound to Si(3)N(4) AFM tips, previously modified with 3-aminopropyl-dimethyl-ethoxysilane, through a homobifunctional poly(ethylene glycol) linker bearing two hydroxysulfosuccinimide esters. The most probable detachment force was found to be 210 pN, when the tip was retracting with a velocity of 1400 nm/s, while the distribution of the detachment distances indicated that the fibrinogen chain can be elongated beyond the length of the physical conformation before detachment. The dependence of the most probable detachment force on the loading rate was examined and the dynamics of fibrinogen binding to the surface were found amenable to the simple expression of the Bell-Evans theory. The theory's expansion, however, by incorporating the concept of the rupture of parallel residue-surface bonds could only describe the detachment of fibrinogen for a small number of such bonds. Finally, the mathematical expression of the Worm-Like Chain model was used to fit the stretching curves before rupture and two interpretations are suggested for the description of the AFM curves with multiple detachment events.

  11. Single-Molecule Analysis of Protein Large-Amplitude Conformational Transitions

    Science.gov (United States)

    Yang, Haw

    2011-03-01

    Proteins have evolved to harness thermal fluctuations, rather than frustrated by them, to carry out chemical transformations and mechanical work. What are, then, the operation and design principles of protein machines? To frame the problem in a tractable way, several basic questions have been formulated to guide the experimental design: (a) How many conformational states can a protein sample on the functionally important timescale? (b) What are the inter-conversion rates between states? (c) How do ligand binding or interactions with other proteins modulate the motions? (d) What are the structural basis of flexibility and its underlying molecular mechanics? Guided by this framework, we have studied protein tyrosine phosphatase B, PtpB, from M. tuberculosis (a virulence factor of tuberculosis and a potential drug target) and adenylate kinase, AK, from E. coli (a ubiquitous energy-balancing enzyme in cells). These domain movements have been followed in real time on their respective catalytic timescales using high-resolution single-molecule Förster resonance energy transfer (FRET) spectroscopy. It is shown quantitatively that both PtpB and AK are capable of dynamically sampling two distinct states that correlate well with those observed by x-ray crystallography. Integrating these microscopic dynamics into macroscopic kinetics allows us to place the experimentally measured free-energy landscape in the context of enzymatic turnovers.

  12. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    Science.gov (United States)

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH.

    Science.gov (United States)

    Kochan, Jakub; Wawro, Mateusz; Kasza, Aneta

    2015-10-01

    Although the concept of combining immunofluorescence (IF) with single-molecule RNA fluorescence in situ hybridization (smRNA FISH) seems obvious, the specific materials used during IF and smRNA FISH make it difficult to perform these procedures simultaneously on the same specimen. Even though there are reports where IF and smRNA FISH were combined with success, these were insufficient in terms of signal intensities, staining patterns, and GFP-compatibility, and a detailed exploration of the various factors that influence IF and smRNA FISH outcome has not been published yet. Here, we report a detailed study of conditions and reagents used in classic IF and smRNA FISH that allowed us to establish an easy, robust, and GFP-compatible procedure. Our protocol enables simultaneous detection of mRNA and protein quantity as well as the subcellular distribution of these molecules in single cells by combining an RNase-free modification of the IF technique and the more recent smRNA FISH method. Using this procedure, we have shown the direct interaction of RNase MCPIP1 with IL-6 mRNA. We also demonstrate the use of our protocol in heterogeneous cell population analysis, revealing cell-to-cell differences in mRNA and protein content.

  14. Compact halo-ligand-conjugated quantum dots for multicolored single-molecule imaging of overcrowding GPCR proteins on cell membranes.

    Science.gov (United States)

    Komatsuzaki, Akihito; Ohyanagi, Tatsuya; Tsukasaki, Yoshikazu; Miyanaga, Yukihiro; Ueda, Masahiro; Jin, Takashi

    2015-03-25

    To detect single molecules within the optical diffraction limit (< ca. 200 nm), a multicolored imaging technique is developed using Halo-ligand conjugated quantum dots (Halo-QDs; <6 nm in diameter). Using three types of Halo-QDs, multicolored single-molecule fluorescence imaging of GPCR proteins in Dictyostelium cells is achieved. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy.

    Science.gov (United States)

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2016-08-23

    Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation. Copyright © 2016 Biophysical Society. All rights reserved.

  16. High-throughput single-molecule force spectroscopy for membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios [M E Mueller Institute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel (Switzerland); Ratera, Merce; Palacin, Manuel [Institute for Research in Biomedicine, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona (Spain); Bippes, Christian A; Mueller, Daniel J [BioTechnology Center, Technical University, Tatzberg 47, D-01307 Dresden (Germany)], E-mail: andreas.engel@unibas.ch, E-mail: dimitrios.fotiadis@mci.unibe.ch

    2008-09-24

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether {approx}400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with {approx}200 (AdiC) and {approx}400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  17. High-throughput single-molecule force spectroscopy for membrane proteins

    International Nuclear Information System (INIS)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios; Ratera, Merce; Palacin, Manuel; Bippes, Christian A; Mueller, Daniel J

    2008-01-01

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ∼400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ∼200 (AdiC) and ∼400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications

  18. An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

    Science.gov (United States)

    Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

    2017-09-22

    Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

  19. Nucleotide excision repair at the single-molecule level : analysis of the E. coli UvrA protein

    NARCIS (Netherlands)

    Wagner, Koen

    2011-01-01

    In this thesis, the characteristics of the Escherichia coli UvrA protein were analyzed with microscopy techniques that allow detection of protein complexes at the single-molecule level. Together with UvrB and UvrC, UvrA catalyzes the excision of damaged DNA from the bacterial genome. This DNA repair

  20. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    Science.gov (United States)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  1. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! Kankan Bhattacharyya. General Article Volume 20 Issue 2 February 2015 pp 151-164. Fulltext. Click here to view fulltext PDF. Permanent link:

  2. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! ... Author Affiliations. Kankan Bhattacharyya1. Department of Physical Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata 700 032 India.

  3. Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA-protein interaction

    International Nuclear Information System (INIS)

    Kurita, Hirofumi; Yasuda, Hachiro; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2009-01-01

    We carried out an individual DNA manipulation using an optical trapping for a microbead. This manipulation system is based on a fluorescent microscopy equipped with an IR laser. Both ends of linear DNA molecule were labeled with a biotin and a thiol group, respectively. Then the biotinylated end was attached to a microbead, and the other was immobilized on a thiol-linkable glass surface. We controlled the form of an individual DNA molecule by moving the focal point of IR laser, which trapped the microbead. In addition, we applied single-molecule approach to analyze DNA hydrolysis. We also used microchannel for single-molecule observation of DNA hydrolysis. The shortening of DNA in length caused by enzymatic hydrolysis was observed in real-time. The single-molecule DNA manipulation should contribute to elucidate detailed mechanisms of DNA-protein interactions

  4. Life in extreme environments: single molecule force spectroscopy as a tool to explore proteins from extremophilic organisms.

    Science.gov (United States)

    Tych, Katarzyna M; Hoffmann, Toni; Batchelor, Matthew; Hughes, Megan L; Kendrick, Katherine E; Walsh, Danielle L; Wilson, Michael; Brockwell, David J; Dougan, Lorna

    2015-04-01

    Extremophiles are organisms which survive and thrive in extreme environments. The proteins from extremophilic single-celled organisms have received considerable attention as they are structurally stable and functionally active under extreme physical and chemical conditions. In this short article, we provide an introduction to extremophiles, the structural adaptations of proteins from extremophilic organisms and the exploitation of these proteins in industrial applications. We provide a review of recent developments which have utilized single molecule force spectroscopy to mechanically manipulate proteins from extremophilic organisms and the information which has been gained about their stability, flexibility and underlying energy landscapes.

  5. Single-molecule nanopore enzymology

    Science.gov (United States)

    Wloka, Carsten; Maglia, Giovanni

    2017-01-01

    Biological nanopores are a class of membrane proteins that open nanoscale water-conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. More recently, proteins and enzymes have started being analysed with nanopores. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here we describe the approaches and challenges in nanopore enzymology. PMID:28630164

  6. Electronic transport in single-helical protein molecules: Effects of multiple charge conduction pathways and helical symmetry

    Energy Technology Data Exchange (ETDEWEB)

    Kundu, Sourav, E-mail: sourav.kunduphy@gmail.com; Karmakar, S.N.

    2016-07-15

    We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of environmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.

  7. Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

    Science.gov (United States)

    Landry, Markita Patricia; Ando, Hiroki; Chen, Allen Y.; Cao, Jicong; Kottadiel, Vishal Isaac; Chio, Linda; Yang, Darwin; Dong, Juyao; Lu, Timothy K.; Strano, Michael S.

    2017-05-01

    A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

  8. Single-Molecule Nanomagnets

    Science.gov (United States)

    Friedman, Jonathan R.; Sarachik, Myriam P.

    2010-04-01

    Single-molecule magnets straddle the classical and quantum mechanical worlds, displaying many fascinating phenomena. They may have important technological applications in information storage and quantum computation. We review the physical properties of two prototypical molecular nanomagnets, Mn12-acetate and Fe8: Each behaves as a rigid, spin-10 object and exhibits tunneling between up and down directions. As temperature is lowered, the spin-reversal process evolves from thermal activation to pure quantum tunneling. At low temperatures, magnetic avalanches occur in which the magnetization of an entire sample rapidly reverses. We discuss the important role that symmetry-breaking fields play in driving tunneling and in producing Berry-phase interference. Recent experimental advances indicate that quantum coherence can be maintained on timescales sufficient to allow a meaningful number of quantum computing operations to be performed. Efforts are under way to create monolayers and to address and manipulate individual molecules.

  9. Control of transmembrane protein diffusion within the postsynaptic density assessed by simultaneous single-molecule tracking and localization microscopy

    Directory of Open Access Journals (Sweden)

    Thomas A Blanpied

    2016-07-01

    Full Text Available Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using uPAINT over PSDs whose internal structure was simultaneously resolved using PALM. The results provide important experimental confirmation that PSD scaffold density protein strongly influences the mobility of transmembrane proteins. Tracking a protein with a cytosolic domain that does not bind PSD-95 still was slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize receptors even in the absence of binding. Because numerous proteins thought to be

  10. A Single-Molecule View of Genome Editing Proteins: Biophysical Mechanisms for TALEs and CRISPR/Cas9.

    Science.gov (United States)

    Cuculis, Luke; Schroeder, Charles M

    2017-06-07

    Exciting new advances in genome engineering have unlocked the potential to radically alter the treatment of human disease. In this review, we discuss the application of single-molecule techniques to uncover the mechanisms behind two premier classes of genome editing proteins: transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas). These technologies have facilitated a striking number of gene editing applications in a variety of organisms; however, we are only beginning to understand the molecular mechanisms governing the DNA editing properties of these systems. Here, we discuss the DNA search and recognition process for TALEs and Cas9 that have been revealed by recent single-molecule experiments.

  11. Lanthanide single molecule magnets

    CERN Document Server

    Tang, Jinkui

    2015-01-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs, and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures – an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and...

  12. Lanthanide single molecule magnets

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jinkui; Zhang, Peng [Chinese Academy of Sciences, Changchun (China). Changchun Inst. of Applied Chemistry

    2015-10-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures - an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and explore new directions.

  13. Single-Molecule Protein Folding: A Study of the Surface-Mediated Conformational Dynamics of a Model Amphipathic Peptide

    Science.gov (United States)

    Cunningham, Joy; English, Douglas

    2004-03-01

    Most surface-active polypeptides, composed of 10-50 amino acids, are devoid of well-defined tertiary structure. The conformation of these proteins is greatly dependent upon their environment and may assume totally different characteristics in an aqueous environment, in a detergent micelle, or in an organic solvent. Most antimicrobial peptides are helix-forming and are activated upon interaction with a membrane-mimicking environment. We are seeking to physically characterize the mechanism of membrane-peptide interaction through studying a simple model peptide, MT-1. MT-1 was designed as a nonhomologous analogue of melittin, the principle component in bee venom. We are using single molecule spectroscopy to examine the induction of secondary structure upon interaction of MT-1 with various membrane-mimicking interfaces. Specifically, we monitor coil-to-helix transition through single molecule fluorescence resonance energy transfer (sm-FRET) to determine conformational distributions of folded and unfolded peptides at an interface. Studies with MT-1 will focus upon the biologically relevant issues of orientation, aggregation, and folding at surfaces using both ensemble and single molecule experiments.

  14. Effect of the HIV-1 nucleocapsid protein on reverse transcriptase pause sites revealed by single molecule microscopy

    Science.gov (United States)

    Jouonang, A.; Przybilla, F.; Godet, J.; Sharma, K. K.; Restlé, T.; de Rocquigny, H.; Darlix, J.-L.; Kenfack, C.; Didier, P.; Mély, Y.

    2013-02-01

    During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into the NCp7 molecular mechanism in reverse transcription.

  15. Quantum transport through single molecules

    NARCIS (Netherlands)

    Osorio Oliveros, E.A.

    2009-01-01

    This thesis describes three-terminal transport measurements through single molecules. The interest in this field stems from the dream that single molecules will form the building blocks for future nanoscale electronic devices. The advantages are their small size -nanometers-, and their synthetic

  16. Single-molecule analysis of ligand efficacy in beta(2)AR-G-protein activation

    DEFF Research Database (Denmark)

    Gregorio, G. Glenn; Masureel, Matthieu; Hilger, Daniel

    2017-01-01

    G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail ...

  17. Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

    International Nuclear Information System (INIS)

    Jiang Dafeng; Liu Chunxia; Wang Lei; Jiang Wei

    2010-01-01

    A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 x 10 -14 -3.0 x 10 -12 mol L -1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

  18. Membrane protein nanoclustering as a functional unit of immune cells : from nanoscopy to single molecule dynamics

    OpenAIRE

    Torreño Piña, Juan Andrés

    2015-01-01

    Premi Extraordinari de Doctorat, promoció 2014-2015. Àmbit de Ciències State-of-the-art biophysical techniques featuring high temporal and spatial resolution have allowed for the first time the direct visualization of individual transmembrane proteins on the cell membrane. These techniques have revealed that a large amount of molecular components of the cell membrane do not organize in a random manner but they rather grouped together forming so-called clusters at the nanoscale. Moreover, t...

  19. Theoretical Investigations Regarding Single Molecules

    DEFF Research Database (Denmark)

    Pedersen, Kim Georg Lind

    Neoclassical Valence Bond Theory, Quantum Transport, Quantum Interference, Kondo Effect, and Electron Pumping. Trap a single organic molecule between two electrodes and apply a bias voltage across this "molecular junction". When electrons pass through the molecule, the different electron paths can...... interfere destructively or constructively. Destructive interference effects in electron transport could potentially improve thermo-electrics, organic logic circuits and energy harvesting. We have investigated destructive interference in off-resonant transport through organic molecules, and have found a set...

  20. Single Molecule Electronics and Devices

    Science.gov (United States)

    Tsutsui, Makusu; Taniguchi, Masateru

    2012-01-01

    The manufacture of integrated circuits with single-molecule building blocks is a goal of molecular electronics. While research in the past has been limited to bulk experiments on self-assembled monolayers, advances in technology have now enabled us to fabricate single-molecule junctions. This has led to significant progress in understanding electron transport in molecular systems at the single-molecule level and the concomitant emergence of new device concepts. Here, we review recent developments in this field. We summarize the methods currently used to form metal-molecule-metal structures and some single-molecule techniques essential for characterizing molecular junctions such as inelastic electron tunnelling spectroscopy. We then highlight several important achievements, including demonstration of single-molecule diodes, transistors, and switches that make use of electrical, photo, and mechanical stimulation to control the electron transport. We also discuss intriguing issues to be addressed further in the future such as heat and thermoelectric transport in an individual molecule. PMID:22969345

  1. Single molecule tracking

    Science.gov (United States)

    Shera, E. Brooks

    1988-01-01

    A detection system is provided for identifying individual particles or molecules having characteristic emission in a flow train of the particles in a flow cell. A position sensitive sensor is located adjacent the flow cell in a position effective to detect the emissions from the particles within the flow cell and to assign spatial and temporal coordinates for the detected emissions. A computer is then enabled to predict spatial and temporal coordinates for the particle in the flow train as a function of a first detected emission. Comparison hardware or software then compares subsequent detected spatial and temporal coordinates with the predicted spatial and temporal coordinates to determine whether subsequently detected emissions originate from a particle in the train of particles. In one embodiment, the particles include fluorescent dyes which are excited to fluoresce a spectrum characteristic of the particular particle. Photones are emitted adjacent at least one microchannel plate sensor to enable spatial and temporal coordinates to be assigned. The effect of comparing detected coordinates with predicted coordinates is to define a moving sample volume which effectively precludes the effects of background emissions.

  2. Trapping and manipulating single molecules of DNA

    Science.gov (United States)

    Shon, Min Ju

    This thesis presents the development and application of nanoscale techniques to trap and manipulate biomolecules, with a focus on DNA. These methods combine single-molecule microscopy and nano- and micro-fabrication to study biophysical properties of DNA and proteins. The Dimple Machine is a lab-on-a-chip device that can isolate and confine a small number of molecules from a bulk solution. It traps molecules in nanofabricated chambers, or "dimples", and the trapped molecules are then studied on a fluorescence microscope at the single-molecule level. The sampling of bulk solution by dimples is representative, reproducible, and automated, enabling highthroughput single-molecule experiments. The device was applied to study hybridization of oligonucleotides, particularly in the context of reaction thermodynamics and kinetics in nanoconfinement. The DNA Pulley is a system to study protein binding and the local mechanical properties of DNA. A molecule of DNA is tethered to a surface on one end, and a superparamagnetic bead is attached to the other. A magnet pulls the DNA taut, and a silicon nitride knife with a nanoscale blade scans the DNA along its contour. Information on the local properties of the DNA is extracted by tracking the bead with nanometer precision in a white-light microscope. The system can detect proteins bound to DNA and localize their recognition sites, as shown with a model protein, EcoRI restriction enzyme. Progress on the measurements of nano-mechanical properties of DNA is included.

  3. A single β-octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of partner protein binding.

    Directory of Open Access Journals (Sweden)

    Mousheng Wu

    Full Text Available The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners. In cell-based fluorescence complementation assays, Nef forms homodimers at the plasma membrane. However, recombinant Nef proteins often exist as monomers in solution, suggesting that membrane interaction may also trigger monomer to dimer transitions. In this study, we show that monomeric Nef core proteins can be induced to form dimers in the presence of low concentrations of the non-ionic surfactant, β-octyl glucoside (βOG. X-ray crystallography revealed that a single βOG molecule is present in the Nef dimer, with the 8-carbon acyl chain of the ligand binding to a hydrophobic pocket formed by the dimer interface. This Nef-βOG dimer interface involves helix αB, as observed in previous dimer structures, as well as a helix formed by N-terminal residues 54-66. Nef dimer formation is stabilized in solution by the addition of βOG, providing biochemical validation for the crystal structure. These observations together suggest that the interaction with host cell lipid mediators or other hydrophobic ligands may play a role in Nef dimerization, which has been previously linked to multiple Nef functions including host cell protein kinase activation, CD4 downregulation, and enhancement of HIV-1 replication.

  4. Single-molecule spectroscopy of LHCSR1 protein dynamics identifies two distinct states responsible for multi-timescale photosynthetic photoprotection

    Science.gov (United States)

    Kondo, Toru; Pinnola, Alberta; Chen, Wei Jia; Dall'Osto, Luca; Bassi, Roberto; Schlau-Cohen, Gabriela S.

    2017-08-01

    In oxygenic photosynthesis, light harvesting is regulated to safely dissipate excess energy and prevent the formation of harmful photoproducts. Regulation is known to be necessary for fitness, but the molecular mechanisms are not understood. One challenge has been that ensemble experiments average over active and dissipative behaviours, preventing identification of distinct states. Here, we use single-molecule spectroscopy to uncover the photoprotective states and dynamics of the light-harvesting complex stress-related 1 (LHCSR1) protein, which is responsible for dissipation in green algae and moss. We discover the existence of two dissipative states. We find that one of these states is activated by pH and the other by carotenoid composition, and that distinct protein dynamics regulate these states. Together, these two states enable the organism to respond to two types of intermittency in solar intensity—step changes (clouds and shadows) and ramp changes (sunrise), respectively. Our findings reveal key control mechanisms underlying photoprotective dissipation, with implications for increasing biomass yields and developing robust solar energy devices.

  5. Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.

    Science.gov (United States)

    Tang, Jilin; Krajcikova, Daniela; Zhu, Rong; Ebner, Andreas; Cutting, Simon; Gruber, Hermann J; Barak, Imrich; Hinterdorfer, Peter

    2007-01-01

    Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores. Copyright (c) 2007 John Wiley & Sons, Ltd.

  6. Nanopore analytics: sensing of single molecules.

    Science.gov (United States)

    Howorka, Stefan; Siwy, Zuzanna

    2009-08-01

    In nanopore analytics, individual molecules pass through a single nanopore giving rise to detectable temporary blockades in ionic pore current. Reflecting its simplicity, nanopore analytics has gained popularity and can be conducted with natural protein as well as man-made polymeric and inorganic pores. The spectrum of detectable analytes ranges from nucleic acids, peptides, proteins, and biomolecular complexes to organic polymers and small molecules. Apart from being an analytical tool, nanopores have developed into a general platform technology to investigate the biophysics, physicochemistry, and chemistry of individual molecules (critical review, 310 references).

  7. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  8. Single molecule Studies of DNA Mismatch Repair

    Science.gov (United States)

    Erie, Dorothy A.; Weninger, Keith R.

    2015-01-01

    DNA mismatch repair involves is a widely conserved set of proteins that is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair. PMID:24746644

  9. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  10. Single-Molecule Imaging of GPCR Interactions.

    Science.gov (United States)

    Calebiro, Davide; Sungkaworn, Titiwat

    2018-02-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of great interest as pharmacological targets. Although the occurrence of GPCR signaling nanodomains has long been hypothesized based on indirect evidence, this and other fundamental aspects of GPCR signaling have been difficult to prove. The advent of single-molecule microscopy methods, which allow direct visualization of individual membrane proteins with unprecedented spatiotemporal resolution, provides unique opportunities to address several of these open questions. Indeed, recent single-molecule studies have revealed that GPCRs and G proteins transiently interact with each other as well as with structural components of the plasma membrane, leading to the formation of dynamic complexes and 'hot spots' for GPCR signaling. Whereas we are only beginning to understand the implications of this unexpected level of complexity, single-molecule approaches are likely to play a crucial role to further dissect the protein-protein interactions that are at the heart of GPCR signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer.

    Science.gov (United States)

    Wollman, Adam J M; Miller, Helen; Zhou, Zhaokun; Leake, Mark C

    2015-04-01

    DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

  12. Single-molecule studies using magnetic traps.

    Science.gov (United States)

    Lionnet, Timothée; Allemand, Jean-François; Revyakin, Andrey; Strick, Terence R; Saleh, Omar A; Bensimon, David; Croquette, Vincent

    2012-01-01

    In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 pN. In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. In this article, we describe the principle of the magnetic trap, as well as its use in the measurement of the elastic properties of DNA and the study of DNA-protein interactions.

  13. Single-molecule magnet engineering

    DEFF Research Database (Denmark)

    Pedersen, Kasper Steen; Bendix, Jesper; Clérac, Rodolphe

    2014-01-01

    Tailoring the specific magnetic properties of any material relies on the topological control of the constituent metal ion building blocks. Although this general approach does not seem to be easily applied to traditional inorganic bulk magnets, coordination chemistry offers a unique tool...... to delicately tune, for instance, the properties of molecules that behave as "magnets", the so-called single-molecule magnets (SMMs). Although many interesting SMMs have been prepared by a more or less serendipitous approach, the assembly of predesigned, isolatable molecular entities into higher nuclearity...... complexes constitutes an elegant and fascinating strategy. This Feature article focuses on the use of building blocks or modules (both terms being used indiscriminately) to direct the structure, and therefore also the magnetic properties, of metal ion complexes exhibiting SMM behaviour. This journal is...

  14. Handbook of Single-Molecule Biophysics

    CERN Document Server

    Hinterdorfer, Peter

    2009-01-01

    The last decade has seen the development of a number of novel biophysical methods that allow the manipulation and study of individual biomolecules. The ability to monitor biological processes at this fundamental level of sensitivity has given rise to an improved understanding of the underlying molecular mechanisms. Through the removal of ensemble averaging, distributions and fluctuations of molecular properties can be characterized, transient intermediates identified, and catalytic mechanisms elucidated. By applying forces on biomolecules while monitoring their activity, important information can be obtained on how proteins couple function to structure. The Handbook of Single-Molecule Biophysics provides an introduction to these techniques and presents an extensive discussion of the new biological insights obtained from them. Coverage includes: Experimental techniques to monitor and manipulate individual biomolecules The use of single-molecule techniques in super-resolution and functional imaging Single-molec...

  15. Single molecule insights on conformational selection and induced fit mechanism

    DEFF Research Database (Denmark)

    Hatzakis, Nikos

    2014-01-01

    of unsynchronized molecules, often masking intrinsic dynamic behavior of proteins and biologically significant transient intermediates. Single molecule measurements are emerging as a powerful tool for characterizing protein function. They offer the direct observation and quantification of the activity, abundance...... and lifetime of multiple states and transient intermediates in the energy landscape, that are typically averaged out in non-synchronized ensemble measurements. Here we survey new insights from single molecule studies that advance our understanding of the molecular mechanisms underlying biomolecular recognition....

  16. Automated imaging system for single molecules

    Science.gov (United States)

    Schwartz, David Charles; Runnheim, Rodney; Forrest, Daniel

    2012-09-18

    There is provided a high throughput automated single molecule image collection and processing system that requires minimal initial user input. The unique features embodied in the present disclosure allow automated collection and initial processing of optical images of single molecules and their assemblies. Correct focus may be automatically maintained while images are collected. Uneven illumination in fluorescence microscopy is accounted for, and an overall robust imaging operation is provided yielding individual images prepared for further processing in external systems. Embodiments described herein are useful in studies of any macromolecules such as DNA, RNA, peptides and proteins. The automated image collection and processing system and method of same may be implemented and deployed over a computer network, and may be ergonomically optimized to facilitate user interaction.

  17. Forces and Kinetics of the Bacillus subtilis Spore Coat Proteins CotY and CotX Binding to CotE Inspected by Single Molecule Force Spectroscopy.

    Science.gov (United States)

    Liu, Huiqing; Krajcikova, Daniela; Wang, Nan; Zhang, Zhe; Wang, Hongda; Barak, Imrich; Tang, Jilin

    2016-02-18

    Spores are uniquely stable cell types that are produced when bacteria encounter nutrient limitations. Spores are encased in a complex multilayered coat, which provides protection against environmental insults. The spore coat of Bacillus subtilis is composed of around 70 individual proteins that are organized into four distinct layers. Here we explored how morphogenetic protein CotE guides formation of the outermost layer of the coat, the crust, around the forespore by focusing on three proteins: CotE, CotY, and CotX. Single molecule force spectroscopy (SMFS) was used to investigate the interactions among CotE, CotY, and CotX at the single-molecule level. Direct interactions among these three proteins were observed. Additionally, the dissociation kinetics was also studied by measuring the unbinding forces of the complexes at different loading rates. A series of kinetic data of these complexes were acquired. It was found that the interaction of CotE and CotY was stronger than that of CotE and CotX.

  18. Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

    Science.gov (United States)

    Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R

    2015-10-22

    One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

  19. Single-Molecule Stochastic Resonance

    Directory of Open Access Journals (Sweden)

    K. Hayashi

    2012-08-01

    Full Text Available Stochastic resonance (SR is a well-known phenomenon in dynamical systems. It consists of the amplification and optimization of the response of a system assisted by stochastic (random or probabilistic noise. Here we carry out the first experimental study of SR in single DNA hairpins which exhibit cooperatively transitions from folded to unfolded configurations under the action of an oscillating mechanical force applied with optical tweezers. By varying the frequency of the force oscillation, we investigate the folding and unfolding kinetics of DNA hairpins in a periodically driven bistable free-energy potential. We measure several SR quantifiers under varied conditions of the experimental setup such as trap stiffness and length of the molecular handles used for single-molecule manipulation. We find that a good quantifier of the SR is the signal-to-noise ratio (SNR of the spectral density of measured fluctuations in molecular extension of the DNA hairpins. The frequency dependence of the SNR exhibits a peak at a frequency value given by the resonance-matching condition. Finally, we carry out experiments on short hairpins that show how SR might be useful for enhancing the detection of conformational molecular transitions of low SNR.

  20. Single particle tracking and single molecule energy transfer

    CERN Document Server

    Bräuchle, Christoph; Michaelis, Jens

    2009-01-01

    Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.

  1. [Dynamics of Irreversible Evaporation of a Water-Protein Droplet and a Problem of Structural and Dynamical Experiments with Single Molecules].

    Science.gov (United States)

    Shaitan, K V; Armeev, G A; Shaytan, A K

    2016-01-01

    We discuss the effect of isothermal and adiabatic evaporation of water on the state of a water-protein droplet. The discussed problem is of current importance due to development of techniques to perform single molecule experiments using free electron lasers. In such structure-dynamic experiments the delivery of a sample into the X-ray beam is performed using the microdroplet injector. The time between the injection and delivery is in the order of microseconds. In this paper we developed a specialized variant of all-atom molecular dynamics simulations for the study of irreversible isothermal evaporation of the droplet. Using in silico experiments we determined the parameters of isothermal evaporation of the water-protein droplet with the sodium and chloride ions in the concentration range of 0.3 M at different temperatures. The energy of irreversible evaporation determined from in silico experiments at the initial stages of evaporation virtually coincides with the specific heat of evaporation for water. For the kinetics of irreversible adiabatic evaporation an exact analytical solution was obtained in the limit of high thermal conductivity of the droplet (or up to the droplet size of -100 Å). This analytical solution incorporates parameters that are determined using in silico. experiments on isothermal droplet evaporation. We show that the kinetics of adiabatic evaporation and cooling of the droplet scales with the droplet size. Our estimates of the water-protemi droplet. freezing rate in the adiabatic regime in a vacuum chamber show that additional techniques for stabilizing the temperature inside the droplet should be used in order to study the conformational transitions of the protein in single molecules. Isothermal and quasi-isothermal conditions are most suitable for studying the conformational transitions upon object functioning. However, in this case it is necessary to take into account the effects of dehydration and rapid increase of ionic strength in an

  2. Spectrally resolved single-molecule electrometry

    Science.gov (United States)

    Ruggeri, F.; Krishnan, M.

    2018-03-01

    Escape-time electrometry is a recently developed experimental technique that offers the ability to measure the effective electrical charge of a single biomolecule in solution with sub-elementary charge precision. The approach relies on measuring the average escape-time of a single charged macromolecule or molecular species transiently confined in an electrostatic fluidic trap. Comparing the experiments with the predictions of a mean-field model of molecular electrostatics, we have found that the measured effective charge even reports on molecular conformation, e.g., folded or disordered state, and non-uniform charge distribution in disordered proteins or polyelectrolytes. Here we demonstrate the ability to use the spectral dimension to distinguish minute differences in electrical charge between individual molecules or molecular species in a single simultaneous measurement, under identical experimental conditions. Using one spectral channel for referenced measurement, this kind of photophysical distinguishability essentially eliminates the need for accurate knowledge of key experimental parameters, otherwise obtained through intensive characterization of the experimental setup. As examples, we demonstrate the ability to detect small differences (˜5%) in the length of double-stranded DNA fragments as well as single amino acid exchange in an intrinsically disordered protein, prothymosin α.

  3. Nano-manipulation of single DNA molecules

    International Nuclear Information System (INIS)

    Hu Jun; Shanghai Jiaotong Univ., Shanghai; Lv Junhong; Wang Guohua; Wang Ying; Li Minqian; Zhang Yi; Li Bin; Li Haikuo; An Hongjie

    2004-01-01

    Nano-manipulation of single atoms and molecules is a critical technique in nanoscience and nanotechnology. This review paper will focus on the recent development of the manipulation of single DNA molecules based on atomic force microscopy (AFM). Precise manipulation has been realized including varied manipulating modes such as 'cutting', 'pushing', 'folding', 'kneading', 'picking up', 'dipping', etc. The cutting accuracy is dominated by the size of the AFM tip, which is usually 10 nm or less. Single DNA fragments can be cut and picked up and then amplified by single molecule PCR. Thus positioning isolation and sequencing can be performed. (authors)

  4. Mechanoenzymatics and Nanoassembly of Single Molecules

    Science.gov (United States)

    Puchner, Elias M.; Gaub, Hermann E.

    We investigated the muscle enzyme, titin kinase, by means of single-molecule force spectroscopy. Our results show that the binding of ATP, which is the first step of its signaling cascade controlling the muscle gene expression and protein turnover, is mechanically induced. The detailed determination of barrier positions in the mechanical activation pathway and the corresponding functional states allow structural insight, by comparing the experiment with molecular dynamics simulations. From our results, we conclude that titin kinase acts as a natural force sensor controlling the muscle build-up. To study the interplay of functional units, we developed the single-molecule cut-and-paste technique, which combines the precision of AFM with the selectivity of DNA hybridization. Functional units can be assembled one-by-one in an arbitrarily predefined pattern, with an accuracy that is better than 11 nm. The cyclic assembly process is optically monitored and mechanically recorded by force-extension traces. Using biotin as a functional unit attached to the transported DNA, patterns of binding sites may be created, to which streptavidin-modified nanoobjects like fluorescent nanoparticles can specifically self-assemble in a second step.

  5. Single Molecule Applications of Quantum Dots

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Elmelund; Jauffred, Liselotte; Brewer, Jonathan R.

    2013-01-01

    Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the single QD sensitivity level. These are described as follows:...... experiments held together with the prospects in localization microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research....

  6. Proteins Are the Body's Worker Molecules

    Science.gov (United States)

    ... Molecule of the Month section of the RCSB Protein Data Bank . (http://www.pdb.org) Molecule of the Month ... helices and beta sheets. Image courtesy of RCSB Protein Data Bank ( http://www.pdb.org ) Click for larger image ...

  7. Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

    Science.gov (United States)

    Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael

    2016-02-01

    Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

  8. Single-molecule nanopore enzymology

    NARCIS (Netherlands)

    Willems, Kherim; Van Meervelt, Veerle; Wloka, Carsten; Maglia, Giovanni

    2017-01-01

    Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor

  9. Single-molecule fluorescence resonance energy transfer shows uniformity in TATA binding protein-induced DNA bending and heterogeneity in bending kinetics.

    Science.gov (United States)

    Blair, Rebecca H; Goodrich, James A; Kugel, Jennifer F

    2012-09-25

    TATA binding protein (TBP) is a key component of the eukaryotic RNA polymerase II transcription machinery that binds to TATA boxes located in the core promoter regions of many genes. Structural and biochemical studies have shown that when TBP binds DNA, it sharply bends the DNA. We used single-molecule fluorescence resonance energy transfer (smFRET) to study DNA bending by human TBP on consensus and mutant TATA boxes in the absence and presence of TFIIA. We found that the state of the bent DNA within populations of TBP-DNA complexes is homogeneous; partially bent intermediates were not observed. In contrast to the results of previous ensemble studies, TBP was found to bend a mutant TATA box to the same extent as the consensus TATA box. Moreover, in the presence of TFIIA, the extent of DNA bending was not significantly changed, although TFIIA did increase the fraction of DNA molecules bound by TBP. Analysis of the kinetics of DNA bending and unbending revealed that on the consensus TATA box two kinetically distinct populations of TBP-DNA complexes exist; however, the bent state of the DNA is the same in the two populations. Our smFRET studies reveal that human TBP bends DNA in a largely uniform manner under a variety of different conditions, which was unexpected given previous ensemble biochemical studies. Our new observations led to us to revise the model for the mechanism of DNA binding by TBP and for how DNA bending is affected by TATA sequence and TFIIA.

  10. Single Molecule Spectroscopy of Electron Transfer

    International Nuclear Information System (INIS)

    Holman, Michael; Zang, Ling; Liu, Ruchuan; Adams, David M.

    2009-01-01

    The objectives of this research are threefold: (1) to develop methods for the study electron transfer processes at the single molecule level, (2) to develop a series of modifiable and structurally well defined molecular and nanoparticle systems suitable for detailed single molecule/particle and bulk spectroscopic investigation, (3) to relate experiment to theory in order to elucidate the dependence of electron transfer processes on molecular and electronic structure, coupling and reorganization energies. We have begun the systematic development of single molecule spectroscopy (SMS) of electron transfer and summaries of recent studies are shown. There is a tremendous need for experiments designed to probe the discrete electronic and molecular dynamic fluctuations of single molecules near electrodes and at nanoparticle surfaces. Single molecule spectroscopy (SMS) has emerged as a powerful method to measure properties of individual molecules which would normally be obscured in ensemble-averaged measurement. Fluctuations in the fluorescence time trajectories contain detailed molecular level statistical and dynamical information of the system. The full distribution of a molecular property is revealed in the stochastic fluctuations, giving information about the range of possible behaviors that lead to the ensemble average. In the case of electron transfer, this level of understanding is particularly important to the field of molecular and nanoscale electronics: from a device-design standpoint, understanding and controlling this picture of the overall range of possible behaviors will likely prove to be as important as designing ia the ideal behavior of any given molecule.

  11. Relaxation dynamics of a single DNA molecule

    Science.gov (United States)

    Goshen, E.; Zhao, W. Z.; Carmon, G.; Rosen, S.; Granek, R.; Feingold, M.

    2005-06-01

    The relaxation of a single DNA molecule is studied. The experimental system consists of optical tweezers and a micron-sized bead that is tethered to the bottom of the sample by a single double-stranded DNA molecule. The bead slows down the DNA relaxation from a strongly stretched configuration such that it is passing through stretched equilibrium states. This allows for a theoretical description of the relaxation trajectory, which is in good agreement with experiment.

  12. Single Molecule Biophysics Experiments and Theory

    CERN Document Server

    Komatsuzaki, Tamiki; Takahashi, Satoshi; Yang, Haw; Silbey, Robert J; Rice, Stuart A; Dinner, Aaron R

    2011-01-01

    Discover the experimental and theoretical developments in optical single-molecule spectroscopy that are changing the ways we think about molecules and atoms The Advances in Chemical Physics series provides the chemical physics field with a forum for critical, authoritative evaluations of advances in every area of the discipline. This latest volume explores the advent of optical single-molecule spectroscopy, and how atomic force microscopy has empowered novel experiments on individual biomolecules, opening up new frontiers in molecular and cell biology and leading to new theoretical approaches

  13. A Brief Introduction to Single-Molecule Fluorescence Methods.

    Science.gov (United States)

    van den Wildenberg, Siet M J L; Prevo, Bram; Peterman, Erwin J G

    2018-01-01

    One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

  14. Single-molecule manipulation and detection.

    Science.gov (United States)

    Zhao, Deyu; Liu, Siyun; Gao, Ying

    2018-01-25

    Compared to conventional ensemble methods, studying macromolecules at single-molecule level can reveal extraordinary clear and even surprising views for a biological reaction. In the past 20 years, single-molecule techniques have been undergoing a very rapid development, and these cutting edge technologies have revolutionized the biological research by facilitating single-molecule manipulation and detection. Here we give a brief review about these advanced techniques, including optical tweezers, magnetic tweezers, atomic force microscopy (AFM), hydrodynamic flow-stretching assay, and single-molecule fluorescence resonance energy transfer (smFRET). We are trying to describe their basic principles and provide a few examples of applications for each technique. This review aims to give a rather introductory survey of single-molecule techniques for audiences with biological or biophysical background. © The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Single-Molecule Interfacial Electron Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Lu, H. Peter [Bowling Green State Univ., Bowling Green, OH (United States). Dept. of Chemistry and Center for Photochemical Sciences

    2017-11-28

    This project is focused on the use of single-molecule high spatial and temporal resolved techniques to study molecular dynamics in condensed phase and at interfaces, especially, the complex reaction dynamics associated with electron and energy transfer rate processes. The complexity and inhomogeneity of the interfacial ET dynamics often present a major challenge for a molecular level comprehension of the intrinsically complex systems, which calls for both higher spatial and temporal resolutions at ultimate single-molecule and single-particle sensitivities. Combined single-molecule spectroscopy and electrochemical atomic force microscopy approaches are unique for heterogeneous and complex interfacial electron transfer systems because the static and dynamic inhomogeneities can be identified and characterized by studying one molecule at a specific nanoscale surface site at a time. The goal of our project is to integrate and apply these spectroscopic imaging and topographic scanning techniques to measure the energy flow and electron flow between molecules and substrate surfaces as a function of surface site geometry and molecular structure. We have been primarily focusing on studying interfacial electron transfer under ambient condition and electrolyte solution involving both single crystal and colloidal TiO2 and related substrates. The resulting molecular level understanding of the fundamental interfacial electron transfer processes will be important for developing efficient light harvesting systems and broadly applicable to problems in fundamental chemistry and physics. We have made significant advancement on deciphering the underlying mechanism of the complex and inhomogeneous interfacial electron transfer dynamics in dyesensitized TiO2 nanoparticle systems that strongly involves with and regulated by molecule-surface interactions. We have studied interfacial electron transfer on TiO2 nanoparticle surfaces by using ultrafast single-molecule

  16. Quantum transport of the single metallocene molecule

    Science.gov (United States)

    Yu, Jing-Xin; Chang, Jing; Wei, Rong-Kai; Liu, Xiu-Ying; Li, Xiao-Dong

    2016-10-01

    The Quantum transport of three single metallocene molecule is investigated by performing theoretical calculations using the non-equilibrium Green's function method combined with density functional theory. We find that the three metallocen molecules structure become stretched along the transport direction, the distance between two Cp rings longer than the other theory and experiment results. The lager conductance is found in nickelocene molecule, the main transmission channel is the electron coupling between molecule and the electrodes is through the Ni dxz and dyz orbitals and the s, dxz, dyz of gold. This is also confirmed by the highest occupied molecular orbital resonance at Fermi level. In addition, negative differential resistance effect is found in the ferrocene, cobaltocene molecules, this is also closely related with the evolution of the transmission spectrum under applied bias.

  17. Single molecule microscopy and spectroscopy: concluding remarks.

    Science.gov (United States)

    van Hulst, Niek F

    2015-01-01

    Chemistry is all about molecules: control, synthesis, interaction and reaction of molecules. All too easily on a blackboard, one draws molecules, their structures and dynamics, to create an insightful picture. The dream is to see these molecules in reality. This is exactly what "Single Molecule Detection" provides: a look at molecules in action at ambient conditions; a breakthrough technology in chemistry, physics and biology. Within the realms of the Royal Society of Chemistry, the Faraday Discussion on "Single Molecule Microscopy and Spectroscopy" was a very appropriate topic for presentation, deliberation and debate. Undoubtedly, the Faraday Discussions have a splendid reputation in stimulating scientific debates along the traditions set by Michael Faraday. Interestingly, back in the 1830's, Faraday himself pursued an experiment that led to the idea that atoms in a compound were joined by an electrical component. He placed two opposite electrodes in a solution of water containing a dissolved compound, and observed that one of the elements of the compound accumulated on one electrode, while the other was deposited on the opposite electrode. Although Faraday was deeply opposed to atomism, he had to recognize that electrical forces were responsible for the joining of atoms. Probably a direct view on the atoms or molecules in his experiment would have convinced him. As such, Michael Faraday might have liked the gathering at Burlington House in September 2015 (). Surely, with the questioning eyes of his bust on the 1st floor corridor, the non-believer Michael Faraday has incited each passer-by to enter into discussion and search for deeper answers at the level of single molecules. In these concluding remarks, highlights of the presented papers and discussions are summarized, complemented by a conclusion on future perspectives.

  18. Single molecule transcription profiling with AFM

    Science.gov (United States)

    Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A.; Gimzewski, James K.

    2007-01-01

    Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations. Based on invited talk at the International Conference on Nanoscience and Technology 2006.

  19. Single molecule transcription profiling with AFM

    International Nuclear Information System (INIS)

    Reed, Jason; Mishra, Bud; Pittenger, Bede; Magonov, Sergei; Troke, Joshua; Teitell, Michael A; Gimzewski, James K

    2007-01-01

    Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from micro-dissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations

  20. Imaging of tautomerism in a single molecule.

    Science.gov (United States)

    Piwoński, Hubert; Stupperich, Clemens; Hartschuh, Achim; Sepioł, Jerzy; Meixner, Alfred; Waluk, Jacek

    2005-04-20

    Fluorescence imaging is used to visualize directly the transfer of two inner hydrogen atoms in single porphycene molecules. This reaction leads to a chemically equivalent but differently oriented structure and hence results in a rotation of the transition dipole moments. By probing single immobilized molecules with an azimuthally polarized laser beam in the focal spot of a confocal microscope we observe ring-like emission patterns, possible only for a chromophore with two nearly orthogonal transition dipole moments. Numerical simulations of the observed emission patterns yield a value of 72 degrees for the angle between the S0-S1 transition moments in the two tautomeric forms.

  1. Regulation of membrane protein function by lipid bilayer elasticity-a single molecule technology to measure the bilayer properties experienced by an embedded protein

    International Nuclear Information System (INIS)

    Lundbaek, Jens August

    2006-01-01

    Membrane protein function is generally regulated by the molecular composition of the host lipid bilayer. The underlying mechanisms have long remained enigmatic. Some cases involve specific molecular interactions, but very often lipids and other amphiphiles, which are adsorbed to lipid bilayers, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes, in the general regulation of membrane protein function, is unclear. This is to a large extent due to lack of a generally accepted framework in which to understand the many observations. The present review summarizes studies which have demonstrated that the hydrophobic interactions between a membrane protein and the host lipid bilayer provide an energetic coupling, whereby protein function can be regulated by the bilayer elasticity. The feasibility of this 'hydrophobic coupling mechanism' has been demonstrated using the gramicidin channel, a model membrane protein, in planar lipid bilayers. Using voltage-dependent sodium channels, N-type calcium channels and GABA A receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic properties experienced by an embedded protein has been developed. A theoretical and technological framework, to study the regulation of membrane protein function by lipid bilayer elasticity, has been established

  2. Regulation of membrane protein function by lipid bilayer elasticity—a single molecule technology to measure the bilayer properties experienced by an embedded protein

    DEFF Research Database (Denmark)

    Lundbæk, Jens August

    2008-01-01

    -dependent sodium channels, N-type calcium channels and GABAA receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic......Membrane protein function is generally regulated by the molecular composition of the host lipid bilayer. The underlying mechanisms have long remained enigmatic. Some cases involve specific molecular interactions, but very often lipids and other amphiphiles, which are adsorbed to lipid bilayers......, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes...

  3. Single Molecule Nanoelectrochemistry in Electrical Junctions.

    Science.gov (United States)

    Nichols, Richard J; Higgins, Simon J

    2016-11-15

    It is now possible to reliably measure single molecule conductance in a wide variety of environments including organic liquids, ultrahigh vacuum, water, ionic liquids, and electrolytes. The most commonly used methods deploy scanning probe microscopes, mechanically formed break junctions, or lithographically formed nanogap contacts. Molecules are generally captured between a pair of facing electrodes, and the junction current response is measured as a function of bias voltage. Gating electrodes can also be added so that the electrostatic potential at the molecular bridge can be independently controlled by this third noncontacting electrode. This can also be achieved in an electrolytic environment using a four-electrode bipotentiostatic configuration, which allows independent electrode potential control of the two contacting electrodes. This is commonly realized using an electrochemical STM and enables single molecule electrical characterization as a function of electrode potential and redox state of the molecular bridge. This has emerged as a powerful tool in modern interfacial electrochemistry and nanoelectrochemistry for studying charge transport across single molecules as a function of electrode potential and the electrolytic environments. Such measurements are possible in electrolytes ranging from aqueous buffers to nonaqueous ionic liquids. In this Account, we illustrate a number of examples of single molecule electrical measurements under electrode potential control use a scanning tunneling microscope (STM) and demonstrate how these can help in the understanding of charge transport in single molecule junctions. Examples showing charge transport following phase coherent tunneling to incoherent charge hopping across redox active molecular bridges are shown. In the case of bipyridinium (or viologen) molecular wires, it is shown how electrochemical reduction leads to an increase of the single molecule conductance, which is controlled by the liquid electrochemical

  4. Fluorescent Biosensors Based on Single-Molecule Counting.

    Science.gov (United States)

    Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

    2016-09-20

    fluorescence signals by specific in vitro/in vivo fluorescent labeling, and consequently, the fluorescent molecules indicate the presence of target molecules. The resultant fluorescence signals may be simply counted by either microfluidic device-integrated confocal microscopy or total internal reflection fluorescence-based single-molecule imaging. We have developed a series of single-molecule counting-based biosensors which can be classified as separation-free and separation-assisted assays. As a proof-of-concept, we demonstrate the applications of single-molecule counting-based biosensors for sensitive detection of various target biomolecules such as DNAs, miRNAs, proteins, enzymes, and intact cells, which may function as the disease-related biomarkers. Moreover, we give a summary of future directions to expand the usability of single-molecule counting-based biosensors including (1) the development of more user-friendly and automated instruments, (2) the discovery of new fluorescent labels and labeling strategies, and (3) the introduction of new concepts for the design of novel biosensors. Due to their high sensitivity, good selectivity, rapidity, and simplicity, we believe that the single-molecule counting-based fluorescent biosensors will indubitably find wide applications in biological research, clinical diagnostics, and drug discovery.

  5. Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

    Science.gov (United States)

    Shirai, Yuki M.; Tsunoyama, Taka A.; Hiramoto-Yamaki, Nao; Hirosawa, Koichiro M.; Shibata, Akihiro C. E.; Kondo, Kenichi; Tsurumune, Atsushi; Ishidate, Fumiyoshi; Kusumi, Akihiro

    2017-01-01

    Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”. PMID:29190677

  6. Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA.

    Science.gov (United States)

    Ren, Hang; Cheyne, Cameron G; Fleming, Aaron M; Burrows, Cynthia J; White, Henry S

    2018-04-18

    Measurement of single-molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. Herein, we report the acid-base titration of a single DNA duplex confined within the wild-type α-hemolysin (α-HL) nanopore for up to 3 h, while monitoring the ionic current through the nanopore. Modulation between two states in the current-time trace for duplexes containing the C:C mismatch in proximity to the latch constriction of α-HL is attributed to the base flipping of the C:C mismatch. As the pH is lowered, the rate for the C:C mismatch to flip from the intra-helical state to the extra-helical state ( k intra-extra ) decreases, while the rate for base flipping from the extra-helical state to the intra-helical state ( k extra-intra ) remains unchanged. Both k intra-extra and k extra-intra are on the order of 1 × 10 -2 s -1 to 1 × 10 -1 s -1 and remain stable over the time scale of the measurement (several hours). Analysis of the pH-dependent kinetics of base flipping using a hidden Markov kinetic model demonstrates that protonation/deprotonation occurs while the base pair is in the intra-helical state. We also demonstrate that the rate of protonation is limited by transport of H + into the α-HL nanopore. Single-molecule kinetic isotope experiments exhibit a large kinetic isotope effect (KIE) for k intra-extra ( k H / k D ≈ 5) but a limited KIE for k extra-intra ( k H / k D ≈ 1.3), supporting our model. Our experiments correspond to the longest single-molecule measurements performed using a nanopore, and demonstrate its application in interrogating mechanisms of single-molecule reactions in confined geometries.

  7. Viruses and Tetraspanins: Lessons from Single Molecule Approaches

    Science.gov (United States)

    Dahmane, Selma; Rubinstein, Eric; Milhiet, Pierre-Emmanuel

    2014-01-01

    Tetraspanins are four-span membrane proteins that are widely distributed in multi-cellular organisms and involved in several infectious diseases. They have the unique property to form a network of protein-protein interaction within the plasma membrane, due to the lateral associations with one another and with other membrane proteins. Tracking tetraspanins at the single molecule level using fluorescence microscopy has revealed the membrane behavior of the tetraspanins CD9 and CD81 in epithelial cell lines, providing a first dynamic view of this network. Single molecule tracking highlighted that these 2 proteins can freely diffuse within the plasma membrane but can also be trapped, permanently or transiently, in tetraspanin-enriched areas. More recently, a similar strategy has been used to investigate tetraspanin membrane behavior in the context of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection. In this review we summarize the main results emphasizing the relationship in terms of membrane partitioning between tetraspanins, some of their partners such as Claudin-1 and EWI-2, and viral proteins during infection. These results will be analyzed in the context of other membrane microdomains, stressing the difference between raft and tetraspanin-enriched microdomains, but also in comparison with virus diffusion at the cell surface. New advanced single molecule techniques that could help to further explore tetraspanin assemblies will be also discussed. PMID:24800676

  8. Experimental and computational characterization of biological liquid crystals: a review of single-molecule bioassays.

    Science.gov (United States)

    Eom, Kilho; Yang, Jaemoon; Park, Jinsung; Yoon, Gwonchan; Soo Sohn, Young; Park, Shinsuk; Yoon, Dae Sung; Na, Sungsoo; Kwon, Taeyun

    2009-09-10

    Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.

  9. Preface: Special Topic on Single-Molecule Biophysics.

    Science.gov (United States)

    Makarov, Dmitrii E; Schuler, Benjamin

    2018-03-28

    Single-molecule measurements are now almost routinely used to study biological systems and processes. The scope of this special topic emphasizes the physics side of single-molecule observations, with the goal of highlighting new developments in physical techniques as well as conceptual insights that single-molecule measurements bring to biophysics. This issue also comprises recent advances in theoretical physical models of single-molecule phenomena, interpretation of single-molecule signals, and fundamental areas of statistical mechanics that are related to single-molecule observations. A particular goal is to illustrate the increasing synergy between theory, simulation, and experiment in single-molecule biophysics.

  10. Single molecule SERS: Perspectives of analytical applications

    Czech Academy of Sciences Publication Activity Database

    Vlčková, B.; Pavel, I.; Sládková, M.; Šišková, K.; Šlouf, Miroslav

    834-836, - (2007), s. 42-47 ISSN 0022-2860. [European Congress on Molecular Spectroscopy /28./. Istanbul, 03.09.2006-08.09.2006] R&D Projects: GA ČR GA203/04/0688 Institutional research plan: CEZ:AV0Z40500505 Keywords : surface-enhanced Raman scattering (SERS) * surface-enhanced resonance Raman (SERRS) * single molecule SERS Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.486, year: 2007

  11. Graphical models for inferring single molecule dynamics

    Directory of Open Access Journals (Sweden)

    Gonzalez Ruben L

    2010-10-01

    Full Text Available Abstract Background The recent explosion of experimental techniques in single molecule biophysics has generated a variety of novel time series data requiring equally novel computational tools for analysis and inference. This article describes in general terms how graphical modeling may be used to learn from biophysical time series data using the variational Bayesian expectation maximization algorithm (VBEM. The discussion is illustrated by the example of single-molecule fluorescence resonance energy transfer (smFRET versus time data, where the smFRET time series is modeled as a hidden Markov model (HMM with Gaussian observables. A detailed description of smFRET is provided as well. Results The VBEM algorithm returns the model’s evidence and an approximating posterior parameter distribution given the data. The former provides a metric for model selection via maximum evidence (ME, and the latter a description of the model’s parameters learned from the data. ME/VBEM provide several advantages over the more commonly used approach of maximum likelihood (ML optimized by the expectation maximization (EM algorithm, the most important being a natural form of model selection and a well-posed (non-divergent optimization problem. Conclusions The results demonstrate the utility of graphical modeling for inference of dynamic processes in single molecule biophysics.

  12. Single-molecule theory of enzymatic inhibition.

    Science.gov (United States)

    Robin, Tal; Reuveni, Shlomi; Urbakh, Michael

    2018-02-22

    The classical theory of enzymatic inhibition takes a deterministic, bulk based approach to quantitatively describe how inhibitors affect the progression of enzymatic reactions. Catalysis at the single-enzyme level is, however, inherently stochastic which could lead to strong deviations from classical predictions. To explore this, we take the single-enzyme perspective and rebuild the theory of enzymatic inhibition from the bottom up. We find that accounting for multi-conformational enzyme structure and intrinsic randomness should strongly change our view on the uncompetitive and mixed modes of inhibition. There, stochastic fluctuations at the single-enzyme level could make inhibitors act as activators; and we state-in terms of experimentally measurable quantities-a mathematical condition for the emergence of this surprising phenomenon. Our findings could explain why certain molecules that inhibit enzymatic activity when substrate concentrations are high, elicit a non-monotonic dose response when substrate concentrations are low.

  13. Single-Molecule Interfacial Electron Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ho, Wilson [Univ. of California, Irvine, CA (United States)

    2018-02-03

    Interfacial electron transfer (ET) plays an important role in many chemical and biological processes. Specifically, interfacial ET in TiO2-based systems is important to solar energy technology, catalysis, and environmental remediation technology. However, the microscopic mechanism of interfacial ET is not well understood with regard to atomic surface structure, molecular structure, bonding, orientation, and motion. In this project, we used two complementary methodologies; single-molecule fluorescence spectroscopy, and scanning-tunneling microscopy and spectroscopy (STM and STS) to address this scientific need. The goal of this project was to integrate these techniques and measure the molecular dependence of ET between adsorbed molecules and TiO2 semiconductor surfaces and the ET induced reactions such as the splitting of water. The scanning probe techniques, STM and STS, are capable of providing the highest spatial resolution but not easily time-resolved data. Single-molecule fluorescence spectroscopy is capable of good time resolution but requires further development to match the spatial resolution of the STM. The integrated approach involving Peter Lu at Bowling Green State University (BGSU) and Wilson Ho at the University of California, Irvine (UC Irvine) produced methods for time and spatially resolved chemical imaging of interfacial electron transfer dynamics and photocatalytic reactions. An integral aspect of the joint research was a significant exchange of graduate students to work at the two institutions. This project bridged complementary approaches to investigate a set of common problems by working with the same molecules on a variety of solid surfaces, but using appropriate techniques to probe under ambient (BGSU) and ultrahigh vacuum (UCI) conditions. The molecular level understanding of the fundamental interfacial electron transfer processes obtained in this joint project will be important for developing efficient light harvesting

  14. Deep learning for single-molecule science

    Science.gov (United States)

    Albrecht, Tim; Slabaugh, Gregory; Alonso, Eduardo; Al-Arif, SM Masudur R.

    2017-10-01

    Exploring and making predictions based on single-molecule data can be challenging, not only due to the sheer size of the datasets, but also because a priori knowledge about the signal characteristics is typically limited and poor signal-to-noise ratio. For example, hypothesis-driven data exploration, informed by an expectation of the signal characteristics, can lead to interpretation bias or loss of information. Equally, even when the different data categories are known, e.g., the four bases in DNA sequencing, it is often difficult to know how to make best use of the available information content. The latest developments in machine learning (ML), so-called deep learning (DL) offer interesting, new avenues to address such challenges. In some applications, such as speech and image recognition, DL has been able to outperform conventional ML strategies and even human performance. However, to date DL has not been applied much in single-molecule science, presumably in part because relatively little is known about the ‘internal workings’ of such DL tools within single-molecule science as a field. In this Tutorial, we make an attempt to illustrate in a step-by-step guide how one of those, a convolutional neural network (CNN), may be used for base calling in DNA sequencing applications. We compare it with a SVM as a more conventional ML method, and discuss some of the strengths and weaknesses of the approach. In particular, a ‘deep’ neural network has many features of a ‘black box’, which has important implications on how we look at and interpret data.

  15. Biological Nanopores: Confined Spaces for Electrochemical Single-Molecule Analysis.

    Science.gov (United States)

    Cao, Chan; Long, Yi-Tao

    2018-02-20

    Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins. In this Account, we highlight recent developments of biological nanopores in DNA-based sensing and in studying the conformational structures of DNA and RNA. Furthermore, we introduce the application of biological nanopores to investigate the conformations of peptides affected by charge, length, and dipole moment and to study disease-related proteins' structures and aggregation transitions influenced by an inhibitor, a promoter, or an applied voltage. To improve the sensing ability of biological nanopores and further extend their application to a wider range of molecular sensing, we focus on exploring novel biological nanopores, such as aerolysin and Stable Protein 1. Aerolysin exhibits an especially high sensitivity for the detection of single oligonucleotides both in current separation and duration. Finally, to facilitate the use of nanopore measurements and statistical analysis

  16. Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Chi, Qijin; Albrecht, Tim

    2005-01-01

    Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class...... tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules...

  17. Hierarchical organization in aggregates of protein molecules

    DEFF Research Database (Denmark)

    Bohr, Henrik; Kyhle, Anders; Sørensen, Alexis Hammer

    1997-01-01

    of the solution and the density of protein are varied shows the existence of specific growth processes resulting in different branch-like structures. The resulting structures are strongly influenced by the shape of each protein molecule. Lysozyme and ribonuclease are found to form spherical structures...

  18. Protein Scaffolding for Small Molecule Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Baker, David [Univ. of Washington, Seattle, WA (United States)

    2014-09-14

    We aim to design hybrid catalysts for energy production and storage that combine the high specificity, affinity, and tunability of proteins with the potent chemical reactivities of small organometallic molecules. The widely used Rosetta and RosettaDesign methodologies will be extended to model novel protein / small molecule catalysts in which one or many small molecule active centers are supported and coordinated by protein scaffolding. The promise of such hybrid molecular systems will be demonstrated with the nickel-phosphine hydrogenase of DuBois et. al.We will enhance the hydrogenase activity of the catalyst by designing protein scaffolds that incorporate proton relays and systematically modulate the local environment of the catalyticcenter. In collaboration with DuBois and Shaw, the designs will be experimentally synthesized and characterized.

  19. Molecular electronics: the single molecule switch and transistor

    NARCIS (Netherlands)

    Sotthewes, Kai; Geskin, Victor; Heimbuch, Rene; Kumar, Avijit; Zandvliet, Henricus J.W.

    2014-01-01

    In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected

  20. Mechanisms of Cellular Proteostasis: Insights from Single-Molecule Approaches

    Science.gov (United States)

    Bustamante, Carlos J.; Kaiser, Christian M.; Maillard, Rodrigo A.; Goldman, Daniel H.; Wilson, Christian A.M.

    2015-01-01

    Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP-dependent proteases. In this review, we describe how applications of single-molecule manipulation methods, in particular optical tweezers, are shedding new light on the molecular mechanisms of quality control during the life cycles of proteins. PMID:24895851

  1. Small molecules targeting heterotrimeric G proteins.

    Science.gov (United States)

    Ayoub, Mohammed Akli

    2018-05-05

    G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors regulating many human and animal physiological functions. Their implication in human pathophysiology is obvious with almost 30-40% medical drugs commercialized today directly targeting GPCRs as molecular entities. However, upon ligand binding GPCRs signal inside the cell through many key signaling, adaptor and regulatory proteins, including various classes of heterotrimeric G proteins. Therefore, G proteins are considered interesting targets for the development of pharmacological tools that are able to modulate their interaction with the receptors, as well as their activation/deactivation processes. In this review, old attempts and recent advances in the development of small molecules that directly target G proteins will be described with an emphasis on their utilization as pharmacological tools to dissect the mechanisms of activation of GPCR-G protein complexes. These molecules constitute a further asset for research in the "hot" areas of GPCR biology, areas such as multiple G protein coupling/signaling, GPCR-G protein preassembly, and GPCR functional selectivity or bias. Moreover, this review gives a particular focus on studies in vitro and in vivo supporting the potential applications of such small molecules in various GPCR/G protein-related diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

    Science.gov (United States)

    2011-01-01

    Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution. PMID:21338175

  3. A brief introduction to single-molecule fluorescence methods

    NARCIS (Netherlands)

    Wildenberg, S.M.J.L.; Prevo, B.; Peterman, E.J.G.; Peterman, EJG; Wuite, GJL

    2011-01-01

    One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow

  4. A brief introduction to single-molecule fluorescence methods

    NARCIS (Netherlands)

    van den Wildenberg, Siet M.J.L.; Prevo, Bram; Peterman, Erwin J.G.

    2018-01-01

    One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also

  5. Magnetization reversal in single molecule magnets

    Science.gov (United States)

    Bokacheva, Louisa

    2002-09-01

    I have studied the magnetization reversal in single molecule magnets (SMMs). SMMs are Van der Waals crystals, consisting of identical molecules containing transition metal ions, with high spin and large uniaxial magnetic anisotropy. They can be considered as ensembles of identical, iso-oriented nanomagnets. At high temperature, these materials behave as superparamagnets and their magnetization reversal occurs by thermal activation. At low temperature they become blocked, and their magnetic relaxation occurs via thermally assisted tunneling or pure quantum tunneling through the anisotropy barrier. We have conducted detailed experimental studies of the magnetization reversal in SMM material Mn12-acetate (Mn12) with S = 10. Low temperature measurements were conducted using micro-Hall effect magnetometry. We performed hysteresis and relaxation studies as a function of temperature, transverse field, and magnetization state of the sample. We identified magnetic sublevels that dominate the tunneling at a given field, temperature and magnetization. We observed a crossover between thermally assisted and pure quantum tunneling. The form of this crossover depends on the magnitude and direction of the applied field. This crossover is abrupt (first-order) and occurs in a narrow temperature interval (tunneling mechanisms in Mn12.

  6. Grafting single molecule magnets on gold nanoparticles.

    Science.gov (United States)

    Perfetti, Mauro; Pineider, Francesco; Poggini, Lorenzo; Otero, Edwige; Mannini, Matteo; Sorace, Lorenzo; Sangregorio, Claudio; Cornia, Andrea; Sessoli, Roberta

    2014-01-29

    The chemical synthesis and characterization of the first hybrid material composed by gold nanoparticles and single molecule magnets (SMMs) are described. Gold nanoparticles are functionalized via ligand exchange using a tetrairon(III) SMM containing two 1,2-dithiolane end groups. The grafting is evidenced by the shift of the plasmon resonance peak recorded with a UV-vis spectrometer, by the suppression of nuclear magnetic resonance signals, by X-ray photoemission spectroscopy peaks, and by transmission electron microscopy images. The latter evidence the formation of aggregates of nanoparticles as a consequence of the cross-linking ability of Fe4 through the two 1,2-dithiolane rings located on opposite sides of the metal core. The presence of intact Fe4 molecules is directly proven by synchrotron-based X-ray absorption spectroscopy and X-ray magnetic circular dichroism spectroscopy, while a detailed magnetic characterization, obtained using electron paramagnetic resonance and alternating-current susceptibility, confirms the persistence of SMM behavior in this new hybrid nanostructure. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

    DEFF Research Database (Denmark)

    Chi, Qijin; Farver, O; Ulstrup, Jens

    2005-01-01

    A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi...... on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on/off current ratio of approximate to 9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative...

  8. Single Molecule Analysis of Resection Tracks.

    Science.gov (United States)

    Huertas, Pablo; Cruz-García, Andrés

    2018-01-01

    Homologous recombination is initiated by the so-called DNA end resection, the 5'-3' nucleolytic degradation of a single strand of the DNA at each side of the break. The presence of resected DNA is an obligatory step for homologous recombination. Moreover, the amount of resected DNA modulates the prevalence of different recombination pathways. In different model organisms, there are several published ways to visualize and measure with more or less detail the amount of DNA resected. In human cells, however, technical constraints hampered the study of resection at high resolution. Some information might be gathered from the study of endonuclease-created DSBs, in which the resection of breaks at known sites can be followed by PCR or ChIP. In this chapter, we describe in detail a novel assay to study DNA end resection in breaks located on unknown positions. Here, we use ionizing radiation to induce double-strand breaks, but the same approach can be used to monitor resection induced by different DNA damaging agents. By modifying the DNA-combing technique, used for high-resolution replication analyses, we can measure resection progression at the level of individual DNA fibers. Thus, we named the method Single Molecule Analysis of Resection Tracks (SMART). We use human cells in culture as a model system, but in principle the same approach would be feasible to any model organism adjusting accordingly the DNA isolation part of the protocol.

  9. Stabilization of protein-protein interaction complexes through small molecules.

    Science.gov (United States)

    Zarzycka, Barbara; Kuenemann, Mélaine A; Miteva, Maria A; Nicolaes, Gerry A F; Vriend, Gert; Sperandio, Olivier

    2016-01-01

    Most of the small molecules that have been identified thus far to modulate protein-protein interactions (PPIs) are inhibitors. Another promising way to interfere with PPI-associated biological processes is to promote PPI stabilization. Even though PPI stabilizers are still scarce, stabilization of PPIs by small molecules is gaining momentum and offers new pharmacological options. Therefore, we have performed a literature survey of PPI stabilization using small molecules. From this, we propose a classification of PPI stabilizers based on their binding mode and the architecture of the complex to facilitate the structure-based design of stabilizers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Watching Single Proteins Using Engineered Nanopores

    Science.gov (United States)

    Movileanu, Liviu

    2014-01-01

    Recent studies in the area of single-molecule detection of proteins with nanopores show a great promise in fundamental science, bionanotechnology and proteomics. In this mini-review, I discuss a comprehensive array of examinations of protein detection and characterization using protein and solid-state nanopores. These investigations demonstrate the power of the single-molecule nanopore measurements to reveal a broad range of functional, structural, biochemical and biophysical features of proteins, such as their backbone flexibility, enzymatic activity, binding affinity as well as their concentration, size and folding state. Engineered nanopores in organic materials and in inorganic membranes coupled with surface modification and protein engineering might provide a new generation of sensing devices for molecular biomedical diagnosis. PMID:24370252

  11. Central dogma at the single-molecule level in living cells.

    Science.gov (United States)

    Li, Gene-Wei; Xie, X Sunney

    2011-07-20

    Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

  12. Small molecule annotation for the Protein Data Bank.

    Science.gov (United States)

    Sen, Sanchayita; Young, Jasmine; Berrisford, John M; Chen, Minyu; Conroy, Matthew J; Dutta, Shuchismita; Di Costanzo, Luigi; Gao, Guanghua; Ghosh, Sutapa; Hudson, Brian P; Igarashi, Reiko; Kengaku, Yumiko; Liang, Yuhe; Peisach, Ezra; Persikova, Irina; Mukhopadhyay, Abhik; Narayanan, Buvaneswari Coimbatore; Sahni, Gaurav; Sato, Junko; Sekharan, Monica; Shao, Chenghua; Tan, Lihua; Zhuravleva, Marina A

    2014-01-01

    The Protein Data Bank (PDB) is the single global repository for three-dimensional structures of biological macromolecules and their complexes, and its more than 100,000 structures contain more than 20,000 distinct ligands or small molecules bound to proteins and nucleic acids. Information about these small molecules and their interactions with proteins and nucleic acids is crucial for our understanding of biochemical processes and vital for structure-based drug design. Small molecules present in a deposited structure may be attached to a polymer or may occur as a separate, non-covalently linked ligand. During curation of a newly deposited structure by wwPDB annotation staff, each molecule is cross-referenced to the PDB Chemical Component Dictionary (CCD). If the molecule is new to the PDB, a dictionary description is created for it. The information about all small molecule components found in the PDB is distributed via the ftp archive as an external reference file. Small molecule annotation in the PDB also includes information about ligand-binding sites and about covalent and other linkages between ligands and macromolecules. During the remediation of the peptide-like antibiotics and inhibitors present in the PDB archive in 2011, it became clear that additional annotation was required for consistent representation of these molecules, which are quite often composed of several sequential subcomponents including modified amino acids and other chemical groups. The connectivity information of the modified amino acids is necessary for correct representation of these biologically interesting molecules. The combined information is made available via a new resource called the Biologically Interesting molecules Reference Dictionary, which is complementary to the CCD and is now routinely used for annotation of peptide-like antibiotics and inhibitors. © The Author(s) 2014. Published by Oxford University Press.

  13. Single Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-coupled Receptor in Living Cells.

    Science.gov (United States)

    Veya, Luc; Piguet, Joachim; Vogel, Horst

    2015-11-13

    Lateral diffusion enables efficient interactions between membrane proteins, leading to signal transmission across the plasma membrane. An open question is how the spatiotemporal distribution of cell surface receptors influences the transmembrane signaling network. Here we addressed this issue by studying the mobility of a prototypical G protein-coupled receptor, the neurokinin-1 receptor, during its different phases of cellular signaling. Attaching a single quantum dot to individual neurokinin-1 receptors enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane. Single receptor trajectories revealed a very heterogeneous mobility distribution pattern with diffusion constants ranging from 0.0005 to 0.1 μm(2)/s comprising receptors freely diffusing and others confined in 100-600-nm-sized membrane domains as well as immobile receptors. A two-dimensional representation of mobility and confinement resolved two major, broadly distributed receptor populations, one showing high mobility and low lateral restriction and the other showing low mobility and high restriction. We found that about 40% of the receptors in the basal state are already confined in membrane domains and are associated with clathrin. After stimulation with an agonist, an additional 30% of receptors became further confined. Using inhibitors of clathrin-mediated endocytosis, we found that the fraction of confined receptors at the basal state depends on the quantity of membrane-associated clathrin and is correlated to a significant decrease of the canonical pathway activity of the receptors. This shows that the high plasticity of receptor mobility is of central importance for receptor homeostasis and fine regulation of receptor activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Single-molecule magnets ``without'' intermolecular interactions

    Science.gov (United States)

    Wernsdorfer, W.; Vergnani, L.; Rodriguez-Douton, M. J.; Cornia, A.; Neugebauer, P.; Barra, A. L.; Sorace, L.; Sessoli, R.

    2012-02-01

    Intermolecular magnetic interactions (dipole-dipole and exchange) affect strongly the magnetic relaxation of crystals of single-molecule magnets (SMMs), especially at low temperature, where quantum tunneling of the magnetization (QTM) dominates. This leads to complex many-body problems [l]. Measurements on magnetically diluted samples are desirable to clearly sort out the behaviour of magnetically-isolated SMMs and to reveal, by comparison, the effect of intermolecular interactions. Here, we diluted a Fe4 SMM into a diamagnetic crystal lattice, affording arrays of independent and iso-oriented magnetic units. We found that the resonant tunnel transitions are much sharper, the tunneling efficiency changes significantly, and two-body QTM transitions disappear. These changes have been rationalized on the basis of a dipolar shuffling mechanism and of transverse dipolar fields, whose effect has been analyzed using a multispin model. Our findings directly prove the impact of intermolecular magnetic couplings on the SMM behaviour and disclose the magnetic response of truly-isolated giant spins in a diamagnetic crystalline environment.[4pt] [1] W. Wernsdorfer, at al, PRL 82, 3903 (1999); PRL 89, 197201 (2002); Nature 416, 406 (2002); IS Tupitsyn, PCE Stamp, NV Prokof'ev, PRB 69, 132406 (2004).

  15. Giant magnetoresistance through a single molecule.

    Science.gov (United States)

    Schmaus, Stefan; Bagrets, Alexei; Nahas, Yasmine; Yamada, Toyo K; Bork, Annika; Bowen, Martin; Beaurepaire, Eric; Evers, Ferdinand; Wulfhekel, Wulf

    2011-03-01

    Magnetoresistance is a change in the resistance of a material system caused by an applied magnetic field. Giant magnetoresistance occurs in structures containing ferromagnetic contacts separated by a metallic non-magnetic spacer, and is now the basis of read heads for hard drives and for new forms of random access memory. Using an insulator (for example, a molecular thin film) rather than a metal as the spacer gives rise to tunnelling magnetoresistance, which typically produces a larger change in resistance for a given magnetic field strength, but also yields higher resistances, which are a disadvantage for real device operation. Here, we demonstrate giant magnetoresistance across a single, non-magnetic hydrogen phthalocyanine molecule contacted by the ferromagnetic tip of a scanning tunnelling microscope. We measure the magnetoresistance to be 60% and the conductance to be 0.26G(0), where G(0) is the quantum of conductance. Theoretical analysis identifies spin-dependent hybridization of molecular and electrode orbitals as the cause of the large magnetoresistance.

  16. Single-Molecule Plasmon Sensing: Current Status and Future Prospects.

    Science.gov (United States)

    Taylor, Adam B; Zijlstra, Peter

    2017-08-25

    Single-molecule detection has long relied on fluorescent labeling with high quantum-yield fluorophores. Plasmon-enhanced detection circumvents the need for labeling by allowing direct optical detection of weakly emitting and completely nonfluorescent species. This review focuses on recent advances in single molecule detection using plasmonic metal nanostructures as a sensing platform, particularly using a single particle-single molecule approach. In the past decade two mechanisms for plasmon-enhanced single-molecule detection have been demonstrated: (1) by plasmonically enhancing the emission of weakly fluorescent biomolecules, or (2) by monitoring shifts of the plasmon resonance induced by single-molecule interactions. We begin with a motivation regarding the importance of single molecule detection, and advantages plasmonic detection offers. We describe both detection mechanisms and discuss challenges and potential solutions. We finalize by highlighting the exciting possibilities in analytical chemistry and medical diagnostics.

  17. Single molecule study of a processivity clamp sliding on DNA

    Energy Technology Data Exchange (ETDEWEB)

    Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A; Barsky, D

    2007-07-05

    Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.

  18. OPE3 : A model system for single-molecule transport

    NARCIS (Netherlands)

    Frisenda, R.

    2016-01-01

    In this dissertation, charge-transport through individual organic molecules is investigated. The single molecules are contacted with two-terminal mechanically controllable break junction gold electrodes and their electrical and mechanical behavior studied at room and low temperature.

  19. Single molecule force spectroscopy: methods and applications in biology

    International Nuclear Information System (INIS)

    Shen Yi; Hu Jun

    2012-01-01

    Single molecule measurements have transformed our view of biomolecules. Owing to the ability of monitoring the activity of individual molecules, we now see them as uniquely structured, fluctuating molecules that stochastically transition between frequently many substrates, as two molecules do not follow precisely the same trajectory. Indeed, it is this discovery of critical yet short-lived substrates that were often missed in ensemble measurements that has perhaps contributed most to the better understanding of biomolecular functioning resulting from single molecule experiments. In this paper, we give a review on the three major techniques of single molecule force spectroscopy, and their applications especially in biology. The single molecular study of biotin-streptavidin interactions is introduced as a successful example. The problems and prospects of the single molecule force spectroscopy are discussed, too. (authors)

  20. Transport through a Single Octanethiol Molecule

    NARCIS (Netherlands)

    Kockmann, D.; Poelsema, Bene; Zandvliet, Henricus J.W.

    2009-01-01

    Octanethiol molecules adsorbed on Pt chains are studied with scanning tunneling microscopy and spectroscopy at 77 K. The head of the octanethiol binds to a Pt atom and the tail is lying flat down on the chain. Open-loop current time traces reveal that the molecule wags its tail and attaches to the

  1. Heterobifunctional crosslinkers for tethering single ligand molecules to scanning probes

    International Nuclear Information System (INIS)

    Riener, Christian K.; Kienberger, Ferry; Hahn, Christoph D.; Buchinger, Gerhard M.; Egwim, Innocent O.C.; Haselgruebler, Thomas; Ebner, Andreas; Romanin, Christoph; Klampfl, Christian; Lackner, Bernd; Prinz, Heino; Blaas, Dieter; Hinterdorfer, Peter; Gruber, Hermann J.

    2003-01-01

    Single molecule recognition force microscopy (SMRFM) is a versatile atomic force microscopy (AFM) method to probe specific interactions of cognitive molecules on the single molecule level. It allows insights to be gained into interaction potentials and kinetic barriers and is capable of mapping interaction sites with nm positional accuracy. These applications require a ligand to be attached to the AFM tip, preferably by a distensible poly(ethylene glycol) (PEG) chain between the measuring tip and the ligand molecule. The PEG chain greatly facilitates specific binding of the ligand to immobile receptor sites on the sample surface. The present study contributes to tip-PEG-ligand tethering in three ways: (i) a convenient synthetic route was found to prepare NH 2 -PEG-COOH which is the key intermediate for long heterobifunctional crosslinkers; (ii) a variety of heterobifunctional PEG derivatives for tip-PEG-ligand linking were prepared from NH 2 -PEG-COOH; (iii) in particular, a new PEG crosslinker with one thiol-reactive end and one terminal nitrilotriacetic acid (NTA) group was synthesized and successfully used to tether His 6 -tagged protein molecules to AFM tips via noncovalent NTA-Ni 2+ -His 6 bridges. The new crosslinker was applied to link a recombinant His 6 -tagged fragment of the very-low density lipoprotein receptor to the AFM tip whereupon specific docking to the capsid of human rhinovirus particles was observed by force microscopy. In a parallel study, the specific interaction of the small GTPase Ran with the nuclear import receptor importin β1 was studied in detail by SMRFM, using the new crosslinker to link His 6 -tagged Ran to the measuring tip [Nat. Struct. Biol. (2003), 10, 553-557

  2. Experimental techniques for single cell and single molecule biomechanics

    International Nuclear Information System (INIS)

    Lim, C.T.; Zhou, E.H.; Li, A.; Vedula, S.R.K.; Fu, H.X.

    2006-01-01

    Stresses and strains that act on the human body can arise either from external physical forces or internal physiological environmental conditions. These biophysical interactions can occur not only at the musculoskeletal but also cellular and molecular levels and can determine the health and function of the human body. Here, we seek to investigate the structure-property-function relationship of cells and biomolecules so as to understand their important physiological functions as well as establish possible connections to human diseases. With the recent advancements in cell and molecular biology, biophysics and nanotechnology, several innovative and state-of-the-art experimental techniques and equipment have been developed to probe the structural and mechanical properties of biostructures from the micro- down to picoscale. Some of these experimental techniques include the optical or laser trap method, micropipette aspiration, step-pressure technique, atomic force microscopy and molecular force spectroscopy. In this article, we will review the basic principles and usage of these techniques to conduct single cell and single molecule biomechanics research

  3. Detecting single DNA molecule interactions with optical microcavities (Presentation Recording)

    Science.gov (United States)

    Vollmer, Frank

    2015-09-01

    as the detection of less than 1 kDa intercalating small molecules[1]. [1] M. D. Baaske, M. R. Foreman, and F. Vollmer, "Single molecule nucleic acid interactions monitored on a label-free microcavity biosensing platform," Nature Nanotechnology, vol. 9, pp. 933-939, 2014. [2] Y. Wu, D. Y. Zhang, P. Yin, and F. Vollmer, "Ultraspecific and Highly Sensitive Nucleic Acid Detection by Integrating a DNA Catalytic Network with a Label-Free Microcavity," Small, vol. 10, pp. 2067-2076, 2014. [3] M. R. Foreman, W.-L. Jin, and F. Vollmer, "Optimizing Detection Limits in Whispering Gallery Mode Biosensing," Optics Express, vol. 22, pp. 5491-5511, 2014. [4] M. A. Santiago-Cordoba, S. V. Boriskina, F. Vollmer, and M. C. Demirel, "Nanoparticle-based protein detection by optical shift of a resonant microcavity," Applied Physics Letters, vol. 99, Aug 2011. [5] M. R. Foreman and F. Vollmer, "Theory of resonance shifts of whispering gallery modes by arbitrary plasmonic nanoparticles," New Journal of Physics, vol. 15, p. 083006, Aug 2013. [6] M. R. Foreman and F. Vollmer "Level repulsion in hybrid photonic-plasmonic microresonators for enhanced biodetection" Phys. Rev. A 88, 023831 (2013).

  4. Compact quantum dots for single-molecule imaging.

    Science.gov (United States)

    Smith, Andrew M; Nie, Shuming

    2012-10-09

    Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology (1-4). To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study. Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation (5). Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large (4,6,7). Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past (8,9). The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an

  5. Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

    International Nuclear Information System (INIS)

    Zhang Jingdong; Chi Qijin; Albrecht, Tim; Kuznetsov, Alexander M.; Grubb, Mikala; Hansen, Allan G.; Wackerbarth, Hainer; Welinder, Anne C.; Ulstrup, Jens

    2005-01-01

    Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class of metallic and semiconductor-based nanoparticles, nano-arrays, nanotubes, and nanopits. Others are based on self-assembled molecular monolayers. The latter extend to bioelectrochemical systems with redox metalloproteins and DNA-based molecules as targets. We overview here some recent achievements in areas of interfacial electrochemical ET systems, mapped to the nanoscale and single-molecule levels. Focus is on both experimental and theoretical studies in our group. Systems addressed are organized monolayers of redox active transition metal complexes, and metalloproteins and metalloenzymes on single-crystal Au(1 1 1)-electrode surfaces. These systems have been investigated by voltammetry, spectroscopy, microcantilever technology, and scanning probe microscopy. A class of Os-complexes has shown suitable as targets for electrochemical in situ scanning tunnelling microscopy (STM), with close to single-molecule scanning tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules have been supported by new theoretical frames, which extend established theory of interfacial electrochemical ET. The electrochemical nanoscale and single-molecule systems discussed are compared with other recent nanoscale and single-molecule systems with conspicuous device-like properties, particularly unimolecular rectifiers and single-molecule transistors. Both of these show analogies to electrochemical in situ STM features of redox molecules and

  6. Electrical and mechanical effects in single-molecule junctions

    NARCIS (Netherlands)

    Seldenthuis, J.S.

    2011-01-01

    In single-molecule junctions, the behavior of a device is determined by the properties of an individual molecule. In this thesis we develop several models to describe both electrical and mechanical effects in such devices, which can be used to design molecules with a specific functionality. We show

  7. Manipulation of organic polyradicals in a single-molecule transistor

    NARCIS (Netherlands)

    Fock, J.; Leijnse, M.; Jennum, K.; Zyazin, A.S.; Paaske, J.; Hedegard, P.; Brondsted Nielsen, M.; Van der Zant, H.S.J.

    2012-01-01

    Inspired by cotunneling spectroscopy of spin-states in a single OPE5-based molecule, we investigate the prospects for electric control of magnetism in purely organic molecules contacted in a three-terminal geometry. Using the gate electrode, the molecule is reversibly switched between three

  8. Atomic-Scale Control of Electron Transport through Single Molecules

    DEFF Research Database (Denmark)

    Wang, Y. F.; Kroger, J.; Berndt, R.

    2010-01-01

    Tin-phthalocyanine molecules adsorbed on Ag(111) were contacted with the tip of a cryogenic scanning tunneling microscope. Orders-of-magnitude variations of the single-molecule junction conductance were achieved by controllably dehydrogenating the molecule and by modifying the atomic structure...

  9. Single Molecule 3D Orientation in Time and Space

    NARCIS (Netherlands)

    Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes; Hübner, Christian G.

    2016-01-01

    Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection

  10. Novel approaches for single molecule activation and detection

    CERN Document Server

    Benfenati, Fabio; Torre, Vincent

    2014-01-01

    How can we obtain tools able to process and exchange information at the molecular scale In order to do this, it is necessary to activate and detect single molecules under controlled conditions. This book focuses on the generation of biologically-inspired molecular devices. These devices are based on the developments of new photonic tools able to activate and stimulate single molecule machines. Additionally, new light sensitive molecules can be selectively activated by photonic tools. These technological innovations will provide a way to control activation of single light-sensitive molecules, a

  11. Single Molecule Studies on Dynamics in Liquid Crystals

    Directory of Open Access Journals (Sweden)

    Daniela Täuber

    2013-09-01

    Full Text Available Single molecule (SM methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC. Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC.

  12. Single molecule studies on dynamics in liquid crystals.

    Science.gov (United States)

    Täuber, Daniela; von Borczyskowski, Christian

    2013-09-26

    Single molecule (SM) methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC). Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC.

  13. Rotation of a single molecule within a supramolecular bearing

    DEFF Research Database (Denmark)

    Gimzewski, J.K.; Joachim, C.; Schlittler, R.R.

    1998-01-01

    Experimental visualization and verification of a single-molecule rotor operating within a supramolecular bearing is reported. Using a scanning tunneling microscope, single molecules were observed to exist in one of two spatially defined states Laterally separated by 0.26 nanometers. One...

  14. Large negative differential conductance in single-molecule break junctions

    NARCIS (Netherlands)

    Perrin, Mickael L.; Frisenda, Riccardo; Koole, Max; Seldenthuis, Johannes S.; Gil, Jose A. Celis; Valkenier, Hennie; Hummelen, Jan C.; Renaud, Nicolas; Grozema, Ferdinand C.; Thijssen, Joseph M.; Dulic, Diana; van der Zant, Herre S. J.

    2014-01-01

    Molecular electronics aims at exploiting the internal structure and electronic orbitals of molecules to construct functional building blocks(1). To date, however, the overwhelming majority of experimentally realized single-molecule junctions can be described as single quantum dots, where transport

  15. Single-molecule probes in organic field-effect transistors

    NARCIS (Netherlands)

    Nicolet, Aurélien Armel Louis

    2007-01-01

    The goal of this thesis is to study charge transport phenomena in organic materials. This is done optically by means of single-molecule spectroscopy in a field-effect transistor based on a molecular crystal. We present (in Chapter 2) a fundamental requirement for single-molecule spectroscopy

  16. A single molecule DNA flow stretching microscope for undergraduates

    NARCIS (Netherlands)

    Williams, Kelly; Grafe, Brendan; Burke, Kathryn M.; Tanner, Nathan; van Oijen, Antoine M.; Loparo, Joseph; Price, Allen C.

    2011-01-01

    The design of a simple, safe, and inexpensive single molecule flow stretching instrument is presented. The instrument uses a low cost upright microscope coupled to a webcam for imaging single DNA molecules that are tethered in an easy to construct microfluidic flow cell. The system requires no

  17. DNA analysis by single molecule stretching in nanofluidic biochips

    DEFF Research Database (Denmark)

    Abad, E.; Juarros, A.; Retolaza, A.

    2011-01-01

    Stretching single DNA molecules by confinement in nanofluidic channels has attracted a great interest during the last few years as a DNA analysis tool. We have designed and fabricated a sealed micro/nanofluidic device for DNA stretching applications, based on the use of the high throughput Nano......Imprint Lithography (NIL) technology combined with a conventional anodic bonding of the silicon base and Pyrex cover. Using this chip, we have performed single molecule imaging on a bench-top fluorescent microscope system. Lambda phage DNA was used as a model sample to characterize the chip. Single molecules of λ...... a method to determining DNA size. The results of this work prove that the developed fabrication process is a good alternative for the fabrication of single molecule DNA biochips and it allows developing a variety of innovative bio/chemical sensors based on single-molecule DNA sequencing devices....

  18. Computer systems for annotation of single molecule fragments

    Science.gov (United States)

    Schwartz, David Charles; Severin, Jessica

    2016-07-19

    There are provided computer systems for visualizing and annotating single molecule images. Annotation systems in accordance with this disclosure allow a user to mark and annotate single molecules of interest and their restriction enzyme cut sites thereby determining the restriction fragments of single nucleic acid molecules. The markings and annotations may be automatically generated by the system in certain embodiments and they may be overlaid translucently onto the single molecule images. An image caching system may be implemented in the computer annotation systems to reduce image processing time. The annotation systems include one or more connectors connecting to one or more databases capable of storing single molecule data as well as other biomedical data. Such diverse array of data can be retrieved and used to validate the markings and annotations. The annotation systems may be implemented and deployed over a computer network. They may be ergonomically optimized to facilitate user interactions.

  19. Controlled transport through a single molecule

    NARCIS (Netherlands)

    Kumar, Avijit; Heimbuch, Rene; Poelsema, Bene; Zandvliet, Henricus J.W.

    2012-01-01

    We demonstrate how an electrode–molecule–electrode junction can be controllably opened and closed by careful tuning of the contacts' interspace and voltage. The molecule, an octanethiol, flips to bridge a ~1 nm interspace between substrate and scanning tunnelling microscope tip when an electric

  20. A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

    NARCIS (Netherlands)

    Moayed, F.; Mashaghi, A.; Tans, S.J.

    2013-01-01

    Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an

  1. Zero-mode waveguide nanophotonic structures for single molecule characterization

    Science.gov (United States)

    Crouch, Garrison M.; Han, Donghoon; Bohn, Paul W.

    2018-05-01

    Single-molecule characterization has become a crucial research tool in the chemical and life sciences, but limitations, such as limited concentration range, inability to control molecular distributions in space, and intrinsic phenomena, such as photobleaching, present significant challenges. Recent developments in non-classical optics and nanophotonics offer promising routes to mitigating these restrictions, such that even low affinity (K D ~ mM) biomolecular interactions can be studied. Here we introduce and review specific nanophotonic devices used to support single molecule studies. Optical nanostructures, such as zero-mode waveguides (ZMWs), are usually fabricated in thin gold or aluminum films and serve to confine the observation volume of optical microspectroscopy to attoliter to zeptoliter volumes. These simple nanostructures allow individual molecules to be isolated for optical and electrochemical analysis, even when the molecules of interest are present at high concentration (µM–mM) in bulk solution. Arrays of ZMWs may be combined with optical probes such as single molecule fluorescence, single molecule fluorescence resonance energy transfer, and fluorescence correlation spectroscopy for distributed analysis of large numbers of single-molecule reactions or binding events in parallel. Furthermore, ZMWs may be used as multifunctional devices, for example by combining optical and electrochemical functions in a single discrete architecture to achieve electrochemical ZMWs. In this review, we will describe the optical properties, fabrication, and applications of ZMWs for single-molecule studies, as well as the integration of ZMWs into systems for chemical and biochemical analysis.

  2. Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging

    OpenAIRE

    Leake, Mark C.; Greene, Nicholas P.; Godun, Rachel M.; Granjon, Thierry; Buchanan, Grant; Chen, Shuyun; Berry, Richard M.; Palmer, Tracy; Berks, Ben C.

    2008-01-01

    The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determi...

  3. Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays

    Directory of Open Access Journals (Sweden)

    Sungsoo Na

    2009-09-01

    Full Text Available Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.

  4. Photothermal cantilever actuation for fast single-molecule force spectroscopy

    Science.gov (United States)

    Stahl, Stefan W.; Puchner, Elias M.; Gaub, Hermann E.

    2009-07-01

    Photothermal cantilever excitation provides a fast and easy to implement means to control the deflection of standard atomic force microscopy cantilevers. Minute heat pulses yield deflections on the order of several tens of nanometers or when the deflection is kept constant, forces of several hundreds of piconewton can be applied. In our case these pulses resulted in less than 1 K temperature changes at the sample position. Here we present and characterize the implementation of photothermal actuation for single-molecule force-spectroscopy experiments. When molecules are stretched under force-clamp conditions, fast control cycles that re-establish the pulling force after the rupture of molecular domains are essential for detecting the complete unfolding pattern with high precision. By combining the fast response of photothermal cantilever excitation with a conventional piezoactuator, a fast force-clamp with high accuracy and large working distances is reached. Simple feedback mechanisms and standard cantilever geometries lead to step response times of less than 90 μs, which is more than one order of magnitude faster than those of conventional force-clamp systems that are based only on piezo feedback. We demonstrate the fast and accurate performance of the setup by unfolding a protein construct consisting of one green fluorescent protein and eight surrounding immunoglobulin domains at constant force.

  5. Controlling single-molecule junction conductance by molecular interactions

    Science.gov (United States)

    Kitaguchi, Y.; Habuka, S.; Okuyama, H.; Hatta, S.; Aruga, T.; Frederiksen, T.; Paulsson, M.; Ueba, H.

    2015-01-01

    For the rational design of single-molecular electronic devices, it is essential to understand environmental effects on the electronic properties of a working molecule. Here we investigate the impact of molecular interactions on the single-molecule conductance by accurately positioning individual molecules on the electrode. To achieve reproducible and precise conductivity measurements, we utilize relatively weak π-bonding between a phenoxy molecule and a STM-tip to form and cleave one contact to the molecule. The anchoring to the other electrode is kept stable using a chalcogen atom with strong bonding to a Cu(110) substrate. These non-destructive measurements permit us to investigate the variation in single-molecule conductance under different but controlled environmental conditions. Combined with density functional theory calculations, we clarify the role of the electrostatic field in the environmental effect that influences the molecular level alignment. PMID:26135251

  6. Precision control of single-molecule electrical junctions.

    Science.gov (United States)

    Haiss, Wolfgang; Wang, Changsheng; Grace, Iain; Batsanov, Andrei S; Schiffrin, David J; Higgins, Simon J; Bryce, Martin R; Lambert, Colin J; Nichols, Richard J

    2006-12-01

    There is much discussion of molecules as components for future electronic devices. However, the contacts, the local environment and the temperature can all affect their electrical properties. This sensitivity, particularly at the single-molecule level, may limit the use of molecules as active electrical components, and therefore it is important to design and evaluate molecular junctions with a robust and stable electrical response over a wide range of junction configurations and temperatures. Here we report an approach to monitor the electrical properties of single-molecule junctions, which involves precise control of the contact spacing and tilt angle of the molecule. Comparison with ab initio transport calculations shows that the tilt-angle dependence of the electrical conductance is a sensitive spectroscopic probe, providing information about the position of the Fermi energy. It is also shown that the electrical properties of flexible molecules are dependent on temperature, whereas those of molecules designed for their rigidity are not.

  7. Direct single-molecule dynamic detection of chemical reactions.

    Science.gov (United States)

    Guan, Jianxin; Jia, Chuancheng; Li, Yanwei; Liu, Zitong; Wang, Jinying; Yang, Zhongyue; Gu, Chunhui; Su, Dingkai; Houk, Kendall N; Zhang, Deqing; Guo, Xuefeng

    2018-02-01

    Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes. For the first time, real-time single-molecule electrical measurements unambiguously show reproducible large-amplitude two-level fluctuations that are highly dependent on solvent environments in a nucleophilic addition reaction of hydroxylamine to a carbonyl group. Both theoretical simulations and ensemble experiments prove that this observation originates from the reversible transition between the reactant and a new intermediate state within a time scale of a few microseconds. These investigations open up a new route that is able to be immediately applied to probe fast single-molecule physics or biophysics with high time resolution, making an important contribution to broad fields beyond reaction chemistry.

  8. Single-molecule tracking in living cells using single quantum dot applications.

    Science.gov (United States)

    Baba, Koichi; Nishida, Kohji

    2012-01-01

    Revealing the behavior of single molecules in living cells is very useful for understanding cellular events. Quantum dot probes are particularly promising tools for revealing how biological events occur at the single molecule level both in vitro and in vivo. In this review, we will introduce how single quantum dot applications are used for single molecule tracking. We will discuss how single quantum dot tracking has been used in several examples of complex biological processes, including membrane dynamics, neuronal function, selective transport mechanisms of the nuclear pore complex, and in vivo real-time observation. We also briefly discuss the prospects for single molecule tracking using advanced probes.

  9. Single-Molecule Electronics: Chemical and Analytical Perspectives.

    Science.gov (United States)

    Nichols, Richard J; Higgins, Simon J

    2015-01-01

    It is now possible to measure the electrical properties of single molecules using a variety of techniques including scanning probe microcopies and mechanically controlled break junctions. Such measurements can be made across a wide range of environments including ambient conditions, organic liquids, ionic liquids, aqueous solutions, electrolytes, and ultra high vacuum. This has given new insights into charge transport across molecule electrical junctions, and these experimental methods have been complemented with increasingly sophisticated theory. This article reviews progress in single-molecule electronics from a chemical perspective and discusses topics such as the molecule-surface coupling in electrical junctions, chemical control, and supramolecular interactions in junctions and gating charge transport. The article concludes with an outlook regarding chemical analysis based on single-molecule conductance.

  10. In situ temperature monitoring in single-molecule FRET experiments

    Science.gov (United States)

    Hartmann, Andreas; Berndt, Frederic; Ollmann, Simon; Krainer, Georg; Schlierf, Michael

    2018-03-01

    Thermodynamic properties of single molecules including enthalpic and entropic contributions are often determined from experiments by a direct control and precise measurement of the local temperature. However, common temperature monitoring techniques using, for example, ultrafine temperature probes can lead to uncertainties as the probe cannot be placed in the vicinity of the molecule of interest. Here, we devised an approach to measure the local temperature in freely diffusing confocal single-molecule Förster Resonance Energy Transfer (smFRET) experiments in situ by directly adding the temperature-sensitive fluorescent dye Rhodamine B, whose fluorescence lifetime serves as a probe of the local temperature in the confocal volume. We demonstrate that the temperature and FRET efficiencies of static and dynamic molecules can be extracted within one measurement simultaneously, without the need of a reference chamber. We anticipate this technique to be particularly useful in the physicochemical analyses of temperature-dependent biomolecular processes from single-molecule measurements.

  11. Single Molecule Raman Detection of Enkephalin on Silver Colloidal Particles

    DEFF Research Database (Denmark)

    Kneipp, Katrin; Kneipp, Holger; Abdali, Salim

    2004-01-01

    the Raman signal the enkephalin molecules have been attached to silver colloidal cluster structures. The experiments demonstrate that the SERS signal of the strongly enhanced ring breathing vibration of phenylalanine at 1000 cm-1 can be used as “intrinsic marker” for detecting a single enkephalin molecule...... and for monitoring its diffusion on the surface of the silver colloidal cluster without using a specific label molecule....

  12. A theoretical justification for single molecule peptide sequencing.

    Directory of Open Access Journals (Sweden)

    Jagannath Swaminathan

    2015-02-01

    Full Text Available The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.

  13. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bin; Xu, Bingqian, E-mail: bxu@engr.uga.edu [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); Lou, Zhichao [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China); Zhang, Haiqian [College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China)

    2016-03-21

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  14. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Science.gov (United States)

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  15. Investigating single molecule adhesion by atomic force spectroscopy.

    Science.gov (United States)

    Stetter, Frank W S; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

    2015-02-27

    Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

  16. Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

    Science.gov (United States)

    Stetter, Frank W. S.; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

    2015-01-01

    Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment. PMID:25867282

  17. Extending Single-Molecule Microscopy Using Optical Fourier Processing

    Science.gov (United States)

    2015-01-01

    This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules. PMID:24745862

  18. Single Molecule Spectroscopy in Chemistry, Physics and Biology Nobel Symposium

    CERN Document Server

    Gräslund, Astrid; Widengren, Jerker

    2010-01-01

    Written by the leading experts in the field, this book describes the development and current state-of-the-art in single molecule spectroscopy. The application of this technique, which started 1989, in physics, chemistry and biosciences is displayed.

  19. Single Molecule Scanning of DNA Radiation Oxidative Damage Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  20. Single-molecule fluorescence microscopy in living Caenorhabditis elegans

    NARCIS (Netherlands)

    van Krugten, Jaap; Peterman, Erwin J.G.

    2018-01-01

    Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal

  1. Single Molecule Scanning of DNA Radiation Oxidative Damage, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  2. Electrochemically-gated single-molecule electrical devices

    International Nuclear Information System (INIS)

    Guo, Shaoyin; Artés, Juan Manuel; Díez-Pérez, Ismael

    2013-01-01

    In the last decade, single-molecule electrical contacts have emerged as a new experimental platform that allows exploring charge transport phenomena in individual molecular blocks. This novel tool has evolved into an essential element within the Molecular Electronics field to understand charge transport processes in hybrid (bio)molecule/electrode interfaces at the nanoscale, and prospect the implementation of active molecular components into functional nanoscale optoelectronic devices. Within this area, three-terminal single-molecule devices have been sought, provided that they are highly desired to achieve full functionality in logic electronic circuits. Despite the latest experimental developments offer consistent methods to bridge a molecule between two electrodes (source and drain in a transistor notation), placing a third electrode (gate) close to the single-molecule electrical contact is still technically challenging. In this vein, electrochemically-gated single-molecule devices have emerged as an experimentally affordable alternative to overcome these technical limitations. In this review, the operating principle of an electrochemically-gated single-molecule device is presented together with the latest experimental methodologies to built them and characterize their charge transport characteristics. Then, an up-to-date comprehensive overview of the most prominent examples will be given, emphasizing on the relationship between the molecular structure and the final device electrical behaviour

  3. Blinking effect and the use of quantum dots in single molecule spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Domingo, M.P. [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Pardo, Julian [Grupo Apoptosis, Inmunidad y Cancer, Departamento Bioquimica y Biologia Molecular y Celular, Fac. Ciencias, Universidad de Zaragoza, Zaragoza (Spain); Fundacion Aragon I-D (ARAID), Gobierno de Aragon, Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain); Graeber, P. [Albert-Ludwigs-Universitaet Freiburg, Institut fuer Physikalische Chemie, Albertstrasse 23a, 79104 Freiburg (Germany); Galvez, E.M., E-mail: eva@icb.csic.es [Instituto de Carboquimica (CSIC), Miguel Luesma 4, 50018 Zaragoza (Spain); Immune Effector Cells Group, Aragon Health Research Institute (IIS Aragon), Biomedical Research Centre of Aragon (CIBA) Fundacion Aragon I-D - ARAID, Gobierno de Aragon, Zaragoza (Spain)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer It is possible to eliminate the blinking effect of a water-soluble QD. Black-Right-Pointing-Pointer We provide a direct method to study protein function and dynamics at the single level. Black-Right-Pointing-Pointer QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the 'on'/'off' states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein-protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

  4. Single-Molecule Spectroscopic Investigations of Amphipathic Helix Formation

    Science.gov (United States)

    Cunningham, Joy Ann; Okamoto, Kenji; English, Douglas

    2004-03-01

    We are using single molecule spectroscopy to examine surface-induced conformational states occurring through interaction of a polypeptide with an interface. Specifically, we investigate the folding of amphipathic helices by using single-molecule fluorescence resonance energy transfer to construct peptide conformational distributions in solution and at interfaces. Analysis of the conformational distributions and kinetics of peptides in different environments reveals properties of the free energy surface for helix formation at an interface relative to formation in solution.

  5. Nanofluidic single-molecule sorting of DNA: a new concept in separation and analysis of biomolecules towards ultimate level performance

    International Nuclear Information System (INIS)

    Yamamoto, Takatoki; Fujii, Teruo

    2010-01-01

    Separation and separation-based analysis of biomolecules are fundamentally important techniques in the field of biotechnology. These techniques, however, depend on stochastic processes that intrinsically involve uncertainty, and thus it is not possible to achieve 100% separation accuracy. Theoretically, the ultimate resolution and sensitivity should be realized in a single-molecule system because of the deterministic nature of single-molecule manipulation. Here, we have proposed and experimentally demonstrated the concept of a 'single-molecule sorter' that detects and correctly identifies individual single molecules, realizing the ultimate level of resolution and sensitivity for any separation-based technology. The single-molecule sorter was created using a nanofluidic network consisting of a single inlet channel that branches off into multiple outlet channels. It includes two major functional elements, namely a single-molecule detection and identification element and a flow path switching element to accurately separate single molecules. With this system we have successfully demonstrated the world's first single-molecule sorting using DNA as a sample molecule. In the future, we hope to expand the application of such devices to comprehensive sorting of single-proteins from a single cell. We also believe that in addition to the single-molecule sorting method reported here, other types of single-molecule based processes will emerge and find use in a wide variety of applications.

  6. Novel Fluorescent Dyes for Single DNA Molecule Techniques

    Directory of Open Access Journals (Sweden)

    Alexander Zarkov

    2013-03-01

    Full Text Available To answer the demands of scientific and medical imaging issues, the family of nucleic acid fluorescent dyes is constantly enlarging. Most of the developed dyes reveal high qualities in bulk solution assays but are inefficient to produce a strong and sufficiently stable signal to enable the application of single-molecule techniques. Therefore, we tested 12 novel monomeric and homodimeric cyanine dyes for potential single DNA molecule imaging. Although their qualities in bulk solutions have already been described, nothing was known about their behavior on a single-molecule level. All 12 dyes demonstrated strong emission when intercalated into single DNA molecules and stretched on a silanized surface, which makes them the perfect choice for fluorescent microscopy imaging. A comparison of their fluorescence intensity and photostability with the most applicable dyes in single-molecule techniques, fluorescent dyes YOYO-1 and POPO-3, was carried out. They all exhibited a strong signal, comparable to that of YOYO-1. However, in contrast to YOYO-1, which is visualized under a green filter only, their emission permits red filter visualization. As their photostability highly exceeds that of similar spectrum POPO-3 dye, the studied dyes stand out as the best choice for a broad range of solid surface single-molecule applications when yellow to red DNA backbone fluorescence is needed.

  7. Electrochemical proton relay at the single-molecule level

    DEFF Research Database (Denmark)

    Kuznetsov, A. M.; Medvedev, I. G.; Ulstrup, Jens

    2009-01-01

    A scheme for the experimental study of single-proton transfer events, based on proton-coupled two-electron transfer between a proton donor and a proton acceptor molecule confined in the tunneling gap between two metal leads in electrolyte solution is suggested. Expressions for the electric current...... are derived and compared with formalism for electron tunneling through redox molecules. The scheme allows studying the kinetics of proton and hydrogen atom transfer as well as kinetic isotope effects at the single-molecule level under electrochemical potential control....

  8. Application of Recognition Tunneling in Single Molecule Identification

    Science.gov (United States)

    Zhao, Yanan

    Single molecule identification is one essential application area of nanotechnology. The application areas including DNA sequencing, peptide sequencing, early disease detection and other industrial applications such as quantitative and quantitative analysis of impurities, etc. The recognition tunneling technique we have developed shows that after functionalization of the probe and substrate of a conventional Scanning Tunneling Microscope with recognition molecules ("tethered molecule-pair" configuration), analyte molecules trapped in the gap that is formed by probe and substrate will bond with the reagent molecules. The stochastic bond formation/breakage fluctuations give insight into the nature of the intermolecular bonding at a single molecule-pair level. The distinct time domain and frequency domain features of tunneling signals were extracted from raw signals of analytes such as amino acids and their enantiomers. The Support Vector Machine (a machine-learning method) was used to do classification and predication based on the signal features generated by analytes, giving over 90% accuracy of separation of up to seven analytes. This opens up a new interface between chemistry and electronics with immediate implications for rapid Peptide/DNA sequencing and molecule identification at single molecule level.

  9. Single Molecule Raman Detection of Enkephalin on Silver Colloidal Particles

    DEFF Research Database (Denmark)

    Kneipp, Katrin; Kneipp, Holger; Abdali, Salim

    2004-01-01

    Enkephalin, an endogeneous substance in the human brain showing morphine-like biological functions, has been detected at the single molecule level based on the surface-enhanced Raman signal of the ring breathing mode of phenylalanine, which is one building block of the molecule. For enhancing...... the Raman signal the enkephalin molecules have been attached to silver colloidal cluster structures. The experiments demonstrate that the SERS signal of the strongly enhanced ring breathing vibration of phenylalanine at 1000 cm-1 can be used as “intrinsic marker” for detecting a single enkephalin molecule...... and for monitoring its diffusion on the surface of the silver colloidal cluster without using a specific label molecule....

  10. Single Molecule Junctions: Probing Contact Chemistry and Fundamental Circuit Laws

    Energy Technology Data Exchange (ETDEWEB)

    Hybertsen M. S.

    2013-04-11

    By exploiting selective link chemistry, formation of single molecule junctions with reproducible conductance has become established. Systematic studies reveal the structure-conductance relationships for diverse molecules. I will draw on experiments from my collaborators at Columbia University, atomic-scale calculations and theory to describe progress in two areas. First, I will describe a novel route to form single molecule junctions, based on SnMe3 terminated molecules, in which gold directly bonds to carbon in the molecule backbone resulting in near ideal contact resistance [1]. Second, comparison of the conductance of junctions formed with molecular species containing either one backbone or two backbones in parallel allows demonstration of the role of quantum interference in the conductance superposition law at the molecular scale [2].

  11. Single transverse mode protein laser

    Science.gov (United States)

    Dogru, Itir Bakis; Min, Kyungtaek; Umar, Muhammad; Bahmani Jalali, Houman; Begar, Efe; Conkar, Deniz; Firat Karalar, Elif Nur; Kim, Sunghwan; Nizamoglu, Sedat

    2017-12-01

    Here, we report a single transverse mode distributed feedback (DFB) protein laser. The gain medium that is composed of enhanced green fluorescent protein in a silk fibroin matrix yields a waveguiding gain layer on a DFB resonator. The thin TiO2 layer on the quartz grating improves optical feedback due to the increased effective refractive index. The protein laser shows a single transverse mode lasing at the wavelength of 520 nm with the threshold level of 92.1 μJ/ mm2.

  12. Fluorescence spectroscopy of single molecules at room temperature and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Taekjip [Univ. of California, Berkeley, CA (United States)

    1996-12-01

    We performed fluorescence spectroscopy of single and pairs of dye molecules on a surface at room temperature. Near field scanning optical microscope (NSOM) and far field scanning optical microscope with multi-color excitation/detection capability were built. The instrument is capable of optical imaging with 100nm resolution and has the sensitivity necessary for single molecule detection. A variety of dynamic events which cannot be observed from an ensemble of molecules is revealed when the molecules are probed one at a time. They include (1) spectral jumps correlated with dark states, (2) individually resolved quantum jumps to and from the meta-stable triplet state, (3) rotational jumps due to desorption/readsorption events of single molecules on the surface. For these studies, a computer controlled optical system which automatically and rapidly locates and performs spectroscopic measurements on single molecules was developed. We also studied the interaction between closely spaced pairs of molecules. In particular, fluorescence resonance energy transfer between a single resonant pair of donor and acceptor molecules was measured. Photodestruction dynamics of the donor or acceptor were used to determine the presence and efficiency of energy transfer Dual molecule spectroscopy was extended to a non-resonant pair of molecules to obtain high resolution differential distance information. By combining NSOM and dual color scheme, we studied the co-localization of parasite proteins and host proteins on a human red blood cell membrane infected with malaria. These dual-molecule techniques can be used to measure distances, relative orientations, and changes in distances/orientations of biological macromolecules with very good spatial, angular and temporal resolutions, hence opening new capabilities in the study of such systems.

  13. Lattice diffusion of a single molecule in solution

    Science.gov (United States)

    Ruggeri, Francesca; Krishnan, Madhavi

    2017-12-01

    The ability to trap a single molecule in an electrostatic potential well in solution has opened up new possibilities for the use of molecular electrical charge to study macromolecular conformation and dynamics at the level of the single entity. Here we study the diffusion of a single macromolecule in a two-dimensional lattice of electrostatic traps in solution. We report the ability to measure both the size and effective electrical charge of a macromolecule by observing single-molecule transport trajectories, typically a few seconds in length, using fluorescence microscopy. While, as shown previously, the time spent by the molecule in a trap is a strong function of its effective charge, we demonstrate here that the average travel time between traps in the landscape yields its hydrodynamic radius. Tailoring the pitch of the lattice thus yields two different experimentally measurable time scales that together uniquely determine both the size and charge of the molecule. Since no information is required on the location of the molecule between consecutive departure and arrival events at lattice sites, the technique is ideally suited to measurements on weakly emitting entities such as single molecules.

  14. Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

    Science.gov (United States)

    Wong, Wesley; Halvorsen, Ken

    2011-03-01

    Precise manipulation of single molecules has led to remarkable insights in physics, chemistry, biology, and medicine. However, two issues that have impeded the widespread adoption of these techniques are equipment cost and the laborious nature of making measurements one molecule at a time. To meet these challenges, we have developed an approach that enables massively parallel single- molecule force measurements using centrifugal force. This approach is realized in the centrifuge force microscope, an instrument in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force- field while their micro-to-nanoscopic motions are observed. We demonstrate high- throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Currently, we are taking steps to integrate high-resolution detection, fluorescence, temperature control and a greater dynamic range in force. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.

  15. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S.A.; Oijen, A.M. van

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules – metabolites, structural proteins, enzymes, oligonucleotides – multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or

  16. Single Molecule Kinetics of ENTH Binding to Lipid Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Rozovsky, Sharon [Univ. of Delaware, Newark, DE (United States); Forstner, Martin B. [Syracuse Univ., NY (United States); Sondermann, Holger [Cornell Univ., Ithaca, NY (United States); Groves, Jay T. [Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2012-04-03

    Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. In this article, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. In addition, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

  17. Blinking effect and the use of quantum dots in single molecule spectroscopy

    International Nuclear Information System (INIS)

    Rombach-Riegraf, Verena; Oswald, Peter; Bienert, Roland; Petersen, Jan; Domingo, M.P.; Pardo, Julian; Gräber, P.; Galvez, E.M.

    2013-01-01

    Highlights: ► It is possible to eliminate the blinking effect of a water-soluble QD. ► We provide a direct method to study protein function and dynamics at the single level. ► QD, potent tool for single molecule studies of biochemical and biological processes. -- Abstract: Luminescent semiconductor nanocrystals (quantum dots, QD) have unique photo-physical properties: high photostability, brightness and narrow size-tunable fluorescence spectra. Due to their unique properties, QD-based single molecule studies have become increasingly more popular during the last years. However QDs show a strong blinking effect (random and intermittent light emission), which may limit their use in single molecule fluorescence studies. QD blinking has been widely studied and some hypotheses have been done to explain this effect. Here we summarise what is known about the blinking effect in QDs, how this phenomenon may affect single molecule studies and, on the other hand, how the “on”/“off” states can be exploited in diverse experimental settings. In addition, we present results showing that site-directed binding of QD to cysteine residues of proteins reduces the blinking effect. This option opens a new possibility of using QDs to study protein–protein interactions and dynamics by single molecule fluorescence without modifying the chemical composition of the solution or the QD surface.

  18. Single-Molecule Electronics with Cross- Conjugated Molecules: Quantum Interference, IETS and Non-Equilibrium "Temperatures"

    DEFF Research Database (Denmark)

    Jørgensen, Jacob Lykkebo

    Abstract The idea of using single-molecules as components in electronic devices is fas- cinating. For this idea to come into fruition, a number of technical and theo- retical challenges must be overcome. In this PhD thesis, the electron-phonon interaction is studied for a special class of molecules......, which is characterised by destructive quantum interference. The molecules are cross-conjugated, which means that the two parts of the molecules are conjugated to a third part, but not to each other. This gives rise to an anti-resonance in the trans- mission. In the low bias and low temperature regime......-conjugated molecules. We nd that the vibrational modes that would be expected to dominate, following the propensity, rules are very weak. Instead, other modes are found to be the dominant ones. We study this phenomenon for a number of cross-conjugated molecules, and link these ndings to the anti...

  19. BRCA Testing by Single-Molecule Molecular Inversion Probes

    NARCIS (Netherlands)

    Neveling, K.; Mensenkamp, A.R.; Derks, R; Kwint, M.P.; Ouchene, H.; Steehouwer, M.; Lier, L.A. van; Bosgoed, E.A.J.; Rikken, A.; Tychon, M.W.J.; Zafeiropoulou, D.; Castelein, S.; Hehir-Kwa, J.Y.; Thung, G.W.; Hofste, T.; Lelieveld, S.H.; Bertens, S.M.; Adan, I.B.; Eijkelenboom, A.; Tops, B.B.J.; Yntema, H.G.; Stokowy, T.; Knappskog, P.M.; Hoberg-Vetti, H.; Steen, V.M.; Boyle, E.; Martin, B.; Ligtenberg, M.J.L.; Shendure, J.; Nelen, M.R.; Hoischen, A.

    2017-01-01

    BACKGROUND: Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the

  20. Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

    Science.gov (United States)

    Schmid, Sonja; Hugel, Thorsten

    2018-03-01

    Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings—such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

  1. Single-Molecule Sensing with Nanopore Confinement: from Chemical Reactions to Biological Interactions.

    Science.gov (United States)

    Lin, Yao; Ying, Yi-Lun; Gao, Rui; Long, Yi-Tao

    2018-03-25

    The nanopore can generate an electrochemical confinement for single-molecule sensing which help understand the fundamental chemical principle in nanoscale dimensions. By observing the generated ionic current, individual bond-making and bond-breaking steps, single biomolecule dynamic conformational changes and electron transfer processes that occur within pore can be monitored with high temporal and current resolution. These single-molecule studies in nanopore confinement are revealing information about the fundamental chemical and biological processes that cannot be extracted from ensemble measurements. In this concept, we introduce and discuss the electrochemical confinement effects on single-molecule covalent reactions, conformational dynamics of individual molecules and host-guest interactions in protein nanopores. Then, we extend the concept of nanopore confinement effects to confine electrochemical redox reactions in solid-state nanopores for developing new sensing mechanisms. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The influence of morphology on excitons in single conjugated molecules

    Science.gov (United States)

    Thiessen, Alexander

    The electronic properties of pi-conjugated molecules are strongly related to their molecular shape and morphology of assembly in three-dimensional space. Understanding the various structure-property relationships is relevant to the applications of these materials in optoelectronic devices such as organic light-emitting diodes, field effect transistors and photovoltaic cells. The fact that conjugated systems interact with visible light opens these materials to a plethora of noninvasive spectroscopic investigation techniques. In this work, electronic properties of different pi-conjugated systems are studied spectroscopically on the ensemble and the single molecule levels. Single molecule spectroscopy is advantageous in that it allows the investigation of the individual nuclear building blocks that contribute to the properties of the ensemble. Additionally, transient photoluminescence spectroscopy methods can provide useful insight into the temporal evolution of the emissive states. In combination with these methods, novel pi-conjugated model molecules are used to probe processes related to exciton dynamics. For the first time, the spatial localization of excited states is probed experimentally in a molecule with a circular chromophoric structure. In addition, a set of model molecules with different geometries is employed to study exciton relaxation in pi-conjugated systems. The molecular morphology is utilized to distinguish between processes such as nuclear reorganization and torsional relaxation. Furthermore, single molecule spectroscopy is used to study the electronic structure of individual polymer chains in the photovoltaic cell material poly-(3-hexylthiophene). Optical spectra of this polymer are known to change with the morphology of the bulk film. Single molecule studies reveal that individual polymer chains exhibit similar behavior and indicate that spectral diversity is an intrinsic property of single P3HT molecules. The main results of this work are the

  3. Fast recognition of single molecules based on single-event photon statistics

    International Nuclear Information System (INIS)

    Dong Shuangli; Huang Tao; Liu Yuan; Wang Jun; Zhang Guofeng; Xiao Liantuan; Jia Suotang

    2007-01-01

    Mandel's Q parameter, which is determined from single-event photon statistics, provides an alternative way to recognize single molecules with fluorescence detection, other than the second-order correlation function. It is shown that the Q parameter of an assumed ideal double-molecule fluorescence with the same average photon number as that of the sample fluorescence can act as the criterion for single-molecule recognition. The influence of signal-to-background ratio and the error estimates for photon statistics are also presented. We have applied this method to ascertain single Cy5 dye molecules within hundreds of milliseconds

  4. Single Molecule Study of DNA Organization and Recombination

    Science.gov (United States)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  5. Challenges for single molecule electronic devices with nanographene and organic molecules. Do single molecules offer potential as elements of electronic devices in the next generation?

    Science.gov (United States)

    Enoki, Toshiaki; Kiguchi, Manabu

    2018-03-01

    Interest in utilizing organic molecules to fabricate electronic materials has existed ever since organic (molecular) semiconductors were first discovered in the 1950s. Since then, scientists have devoted serious effort to the creation of various molecule-based electronic systems, such as molecular metals and molecular superconductors. Single-molecule electronics and the associated basic science have emerged over the past two decades and provided hope for the development of highly integrated molecule-based electronic devices in the future (after the Si-based technology era has ended). Here, nanographenes (nano-sized graphene) with atomically precise structures are among the most promising molecules that can be utilized for electronic/spintronic devices. To manipulate single small molecules for an electronic device, a single molecular junction has been developed. It is a powerful tool that allows even small molecules to be utilized. External electric, magnetic, chemical, and mechanical perturbations can change the physical and chemical properties of molecules in a way that is different from bulk materials. Therefore, the various functionalities of molecules, along with changes induced by external perturbations, allows us to create electronic devices that we cannot create using current top-down Si-based technology. Future challenges that involve the incorporation of condensed matter physics, quantum chemistry calculations, organic synthetic chemistry, and electronic device engineering are expected to open a new era in single-molecule device electronic technology.

  6. Electrochemical single-molecule conductivity of duplex and quadruplex DNA

    DEFF Research Database (Denmark)

    Zhang, Ling; Zhang, Jingdong; Ulstrup, Jens

    2017-01-01

    Photoinduced and electrochemical charge transport in DNA (oligonucleotides, OGNs) and the notions “hopping”, superexchange, polaron, and vibrationally gated charge transport have been in focus over more than two decades. In recent years mapping of electrochemical charge transport of pure and redox...... marked single- and double-strand OGNs has reached the single-molecule level based i.a. on electrochemical in situ scanning tunnelling microscopy (STM) and break-junction (B-J) STM. There are much fewer such reports on “non-canonical” OGN structures such as G-quadruplexes. We discuss first single......-molecule electrochemical conductivity of pure and redox marked duplex OGNs, and address next electrochemistry and electrochemical conductivity in the few reported monolayer and single-molecule G-quadruplex studies. Facile electrochemical electron transfer of iron protoporphyrin IX stacked onto three-quartet 12-guanine...

  7. Monofunctional stealth nanoparticle for unbiased single molecule tracking inside living cells.

    Science.gov (United States)

    Lisse, Domenik; Richter, Christian P; Drees, Christoph; Birkholz, Oliver; You, Changjiang; Rampazzo, Enrico; Piehler, Jacob

    2014-01-01

    On the basis of a protein cage scaffold, we have systematically explored intracellular application of nanoparticles for single molecule studies and discovered that recognition by the autophagy machinery plays a key role for rapid metabolism in the cytosol. Intracellular stealth nanoparticles were achieved by heavy surface PEGylation. By combination with a generic approach for nanoparticle monofunctionalization, efficient labeling of intracellular proteins with high fidelity was accomplished, allowing unbiased long-term tracking of proteins in the outer mitochondrial membrane.

  8. A general approach to break the concentration barrier in single-molecule imaging

    KAUST Repository

    Loveland, Anna B.

    2012-09-09

    Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule\\'s signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations. © 2012 Nature America, Inc. All rights reserved.

  9. 2012 Gordon Research Conference, Single molecule approaches to biology, July 15-20 2012

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez, Julio M. [Columbia Univ., New York, NY (United States)

    2012-04-20

    Single molecule techniques are rapidly occupying a central role in biological research at all levels. This transition was made possible by the availability and dissemination of robust techniques that use fluorescence and force probes to track the conformation of molecules one at a time, in vitro as well as in live cells. Single-molecule approaches have changed the way many biological problems are studied. These novel techniques provide previously unobtainable data on fundamental biochemical processes that are essential for all forms of life. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of the molecular systems that underpin the functioning of living cells. Hence, our conference seeks to disseminate the implementation and use of single molecule techniques in the pursuit of new biological knowledge. Topics covered include: Molecular Motors on the Move; Origin And Fate Of Proteins; Physical Principles Of Life; Molecules and Super-resolution Microscopy; Nanoswitches In Action; Active Motion Or Random Diffusion?; Building Blocks Of Living Cells; From Molecular Mechanics To Physiology; Tug-of-war: Force Spectroscopy Of Single Proteins.

  10. Efficient unfolding pattern recognition in single molecule force spectroscopy data

    Directory of Open Access Journals (Sweden)

    Labudde Dirk

    2011-06-01

    Full Text Available Abstract Background Single-molecule force spectroscopy (SMFS is a technique that measures the force necessary to unfold a protein. SMFS experiments generate Force-Distance (F-D curves. A statistical analysis of a set of F-D curves reveals different unfolding pathways. Information on protein structure, conformation, functional states, and inter- and intra-molecular interactions can be derived. Results In the present work, we propose a pattern recognition algorithm and apply our algorithm to datasets from SMFS experiments on the membrane protein bacterioRhodopsin (bR. We discuss the unfolding pathways found in bR, which are characterised by main peaks and side peaks. A main peak is the result of the pairwise unfolding of the transmembrane helices. In contrast, a side peak is an unfolding event in the alpha-helix or other secondary structural element. The algorithm is capable of detecting side peaks along with main peaks. Therefore, we can detect the individual unfolding pathway as the sequence of events labeled with their occurrences and co-occurrences special to bR's unfolding pathway. We find that side peaks do not co-occur with one another in curves as frequently as main peaks do, which may imply a synergistic effect occurring between helices. While main peaks co-occur as pairs in at least 50% of curves, the side peaks co-occur with one another in less than 10% of curves. Moreover, the algorithm runtime scales well as the dataset size increases. Conclusions Our algorithm satisfies the requirements of an automated methodology that combines high accuracy with efficiency in analyzing SMFS datasets. The algorithm tackles the force spectroscopy analysis bottleneck leading to more consistent and reproducible results.

  11. Rationale for single molecule detection by means of Raman spectroscopy

    International Nuclear Information System (INIS)

    Gaponenko, S.V.; Guzatov, D.V.

    2009-01-01

    A consistent quantum electrodynamical description is proposed of Raman scattering of light by a molecule in a medium with a modified photon density of states. Enhanced local density of states near a metal nanobody is shown to increase a scattering rate by several orders of magnitude, thus providing a rationale for experimental detection of single molecules by means of Raman spectroscopy. For an ellipsoidal particle 10 14 -fold enhancement of the Raman scattering cross-section is obtained. (authors)

  12. Electron transfer dynamics of bistable single-molecule junctions

    DEFF Research Database (Denmark)

    Danilov, A.V; Kubatkin, S.; Kafanov, S. G.

    2006-01-01

    We present transport measurements of single-molecule junctions bridged by a molecule with three benzene rings connected by two double bonds and with thiol end-groups that allow chemical binding to gold electrodes. The I-V curves show switching behavior between two distinct states. By statistical ...... analysis of the switching events, we show that a 300 meV mode mediates the transition between the two states. We propose that breaking and reformation of a S-H bond in the contact zone between molecule and electrode explains the observed bistability....

  13. Single Molecule Study of Photoconversion and Spectral Heterogeneities of Fluorophores

    DEFF Research Database (Denmark)

    Liao, Zhiyu

    of conformational changes and dynamics. The photophysical properties of organic dyes directly determine the quality of the experiments. So the better understanding of the photophysical properties of organic dyes, the better we are able to design the experiments and interpret the data, especially in single molecule...... 104 single molecule measurements. A simple and practical method is introduced to study the characteristics of the photoproducts at the ensemble level. Control experiments reveal that the reaction leading to photobleaching is oxygen related, but the composition of the photoproducts remains inconclusive...... stimulate new pathways in engineering and designing photoconvertible fluorophores, based on the reaction with oxygen or other chemicals. Besides, this results show that dyes that convert into other emissive species could give problems when interpreting single molecule FRET systems. The revealed mechanism...

  14. Coherent interaction of single molecules and plasmonic nanowires

    Science.gov (United States)

    Gerhardt, Ilja; Grotz, Bernhard; Siyushev, Petr; Wrachtrup, Jörg

    2017-09-01

    Quantum plasmonics opens the option to integrate complex quantum optical circuitry onto chip scale devices. In the past, often external light sources were used and nonclassical light was coupled in and out of plasmonic structures, such as hole arrays or waveguide structures. Another option to launch single plasmonic excitations is the coupling of single emitters in the direct proximity of, e.g., a silver or gold nanostructure. Here, we present our attempts to integrate the research of single emitters with wet-chemically grown silver nanowires. The emitters of choice are single organic dye molecules under cryogenic conditions, which are known to act as high-brightness and extremely narrow-band single photon sources. Another advantage is their high optical nonlinearity, such that they might mediate photon-photon interactions on the nanoscale. We report on the coupling of a single molecule fluorescence emission through the wire over the length of several wavelengths. The transmission of coherently emitted photons is proven by an extinction type experiment. As for influencing the spectral properties of a single emitter, we are able to show a remote change of the line-width of a single terrylene molecule, which is in close proximity to the nanowire.

  15. DNA origami as biocompatible surface to match single-molecule and ensemble experiments

    Science.gov (United States)

    Gietl, Andreas; Holzmeister, Phil; Grohmann, Dina; Tinnefeld, Philip

    2012-01-01

    Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements. PMID:22523083

  16. Single-molecule imaging towards precise detection of individual photophysics

    International Nuclear Information System (INIS)

    Tani, Toshiro; Oda, Masaru; Mashimo, Kei; Tachibana, Fumi; Horiuchi, Hiromi

    2006-01-01

    We present our recent study of single fluorescent molecules with specific structure, i.e. tetramethylrhodamine derivative linked with a propyl chain onto silica glass surface. For fluorescent reagent in its synthesis, we used a mixture of two kinds of isomers, which provides a sample with single molecules photophysically different each other even if chemically the same. The isomeric structural difference so introduced in the molecules will provide rather small but probably distinctive photophysical difference, for example, in non-radiative relaxation rates, which we try to detect out with our improved single-molecule microscope imaging technique. To make clear the detectability of such weak inter- or intra-molecular interactions microscopically is significant for versatile applications of single-molecule detections in life science. Our present observation at room temperatures shows so far that such decoupled contributions can be discriminated in the histograms of the intensities of the observed fluorescent spots as broader but separated multi-component structures in the distribution under specific experimental configurations. We will discuss some of the prerequisite for such detections; suitable spatio-temporal resolutions with sufficient S/N ratio, algorithms for data analysis, etc. but also precise sample operations are inevitable

  17. SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY

    Energy Technology Data Exchange (ETDEWEB)

    Professor William Moerner

    2010-07-09

    The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of biomolecular machines: what they do, how they work individually, how they work together, and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site in

  18. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    Science.gov (United States)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  19. Electrochemical Single-Molecule Transistors with Optimized Gate Coupling

    DEFF Research Database (Denmark)

    Osorio, Henrry M.; Catarelli, Samantha; Cea, Pilar

    2015-01-01

    . These data are rationalized in terms of a two-step electrochemical model for charge transport across the redox bridge. In this model the gate coupling in the ionic liquid is found to be fully effective with a modeled gate coupling parameter, ξ, of unity. This compares to a much lower gate coupling parameter......Electrochemical gating at the single molecule level of viologen molecular bridges in ionic liquids is examined. Contrary to previous data recorded in aqueous electrolytes, a clear and sharp peak in the single molecule conductance versus electrochemical potential data is obtained in ionic liquids...

  20. DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis

    Science.gov (United States)

    Wang, Hui; Dunning, James E.; Huang, Albert P.-H.; Nyamwanda, Jacqueline A.; Branton, Daniel

    2004-09-01

    Broad-spectrum analysis of DNA and RNA samples is of increasing importance in the growing field of biotechnology. We show that nanopore measurements may be used to assess the purity, phosphorylation state, and chemical integrity of nucleic acid preparations. In contrast with gel electrophoresis and mass spectrometry, an unprecedented dynamic range of DNA sizes and concentrations can be evaluated in a single data acquisition process that spans minutes. Because the molecule information is quantized and digitally recorded with single-molecule resolution, the sensitivity of the system can be adjusted in real time to detect trace amounts of a particular DNA species.

  1. A single molecule study of cellulase hydrolysis of crystalline cellulose

    Science.gov (United States)

    Liu, Yu-San; Luo, Yonghua; Baker, John O.; Zeng, Yining; Himmel, Michael E.; Smith, Steve; Ding, Shi-You

    2010-02-01

    Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

  2. Single-molecule fluorescence microscopy review: shedding new light on old problems.

    Science.gov (United States)

    Shashkova, Sviatlana; Leake, Mark C

    2017-08-31

    Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called 'green revolution', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called 'super-resolution' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. © 2017 The Author(s).

  3. Towards single molecule biosensors using super-resolution fluorescence microscopy.

    Science.gov (United States)

    Lu, Xun; Nicovich, Philip R; Gaus, Katharina; Gooding, J Justin

    2017-07-15

    Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. A new microcavity design for single molecule detection

    International Nuclear Information System (INIS)

    Steiner, M.; Schleifenbaum, F.; Stupperich, C.; Failla, A.V.; Hartschuh, A.; Meixner, A.J.

    2006-01-01

    We present a new microcavity design which allows for efficient detection of single molecules by measuring the molecular fluorescence emission coupled into a resonant cavity mode. The Fabry-Perot-type microresonator consists of two silver mirrors separated by a thin polymer film doped with dye molecules in ultralow concenctration. By slightly tilting one of the mirrors different cavity lengths can be selected within the same sample. Locally, on a μm scale, the microcavity still acts as a planar Fabry-Perot resonator. Using scanning confocal fluorescence microscopy, single emitters on resonance with a single mode of the microresonator can be spatially addressed. Our microcavity is demonstrated to be well-suited for investigating the coupling mechanism between single quantum emitters and single modes of the electromagnetic field. The microcavity layout could be integrated in a lab-on-a-microchip design for ultrasensitive microfluidic analytics and can be considered as an important improvement for single photon sources based on single molecules operating at room temperature

  5. Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).

    Science.gov (United States)

    Braun, Joerg E; Serebrov, Victor

    2017-01-01

    Recent development of single-molecule techniques to study pre-mRNA splicing has provided insights into the dynamic nature of the spliceosome. Colocalization single-molecule spectroscopy (CoSMoS) allows following spliceosome assembly in real time at single-molecule resolution in the full complexity of cellular extracts. A detailed protocol of CoSMoS has been published previously (Anderson and Hoskins, Methods Mol Biol 1126:217-241, 2014). Here, we provide an update on the technical advances since the first CoSMoS studies including slide surface treatment, data processing, and representation. We describe various labeling strategies to generate RNA reporters with multiple dyes (or other moieties) at specific locations.

  6. Single molecule studies of DNA packaging by bacteriophages

    Science.gov (United States)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages φ29, gamma, and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the φ29 and T4 studies. In the studies of φ29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of lambda and T4 served as systems for comparative studies with φ29. Each system showed similarities to the φ29 system and unique differences. Both the lambda and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to φ29. However, dynamic structural transitions were observed with lambda that are not observed with φ29. The packaging of the lambda genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, lambda and T4 had average packaging velocities under minimal load of 600

  7. A Single Molecule Investigation of the Photostability of Quantum Dots

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Kulatunga, Pasad; Lagerholm, B. Christoffer

    2012-01-01

    Quantum dots (QDs) are very attractive probes for multi-color fluorescence applications. We report here however that single QDs that are subject to continuous blue excitation from a 100W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a per...... is especially detrimental for multi-color single molecule applications, as we regularly observe spectral blue-shifts of 50 nm, or more even after only ten seconds of illumination....

  8. Single-molecule imaging and manipulation of biomolecular machines and systems.

    Science.gov (United States)

    Iino, Ryota; Iida, Tatsuya; Nakamura, Akihiko; Saita, Ei-Ichiro; You, Huijuan; Sako, Yasushi

    2018-02-01

    Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Single molecule DNA detection with an atomic vapor notch filter

    Energy Technology Data Exchange (ETDEWEB)

    Uhland, Denis; Rendler, Torsten; Widmann, Matthias; Lee, Sang-Yun [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Wrachtrup, Joerg; Gerhardt, Ilja [University of Stuttgart and Stuttgart Research Center of Photonic Engineering (SCoPE) and IQST, 3rd Physics Institute, Stuttgart (Germany); Max Planck Institute for Solid State Research, Stuttgart (Germany)

    2015-12-01

    The detection of single molecules has facilitated many advances in life- and material-science. Commonly the fluorescence of dye molecules is detected, which are attached to a non-fluorescent structure under study. For fluorescence microscopy one desires to maximize the detection efficiency together with an efficient suppression of undesired laser leakage. Here we present the use of the narrow-band filtering properties of hot atomic sodium vapor to selectively filter the excitation light from the red-shifted fluorescence of dye labeled single-stranded DNA molecules. A statistical analysis proves an enhancement in detection efficiency of more than 15% in a confocal and in a wide-field configuration. (orig.)

  10. Force-induced tautomerization in a single molecule.

    Science.gov (United States)

    Ladenthin, Janina N; Frederiksen, Thomas; Persson, Mats; Sharp, John C; Gawinkowski, Sylwester; Waluk, Jacek; Kumagai, Takashi

    2016-10-01

    Heat transfer, electrical potential and light energy are common ways to activate chemical reactions. Applied force is another way, but dedicated studies for such a mechanical activation are limited, and this activation is poorly understood at the single-molecule level. Here, we report force-induced tautomerization in a single porphycene molecule on a Cu(110) surface at 5 K, which is studied by scanning probe microscopy and density functional theory calculations. Force spectroscopy quantifies the force needed to trigger tautomerization with submolecular spatial resolution. The calculations show how the reaction pathway and barrier of tautomerization are modified in the presence of a copper tip and reveal the atomistic origin of the process. Moreover, we demonstrate that a chemically inert tip whose apex is terminated by a xenon atom cannot induce the reaction because of a weak interaction with porphycene and a strong relaxation of xenon on the tip as contact to the molecule is formed.

  11. Single-molecule studies of the twisted, knotted, and broken genome

    NARCIS (Netherlands)

    Van Loenhout, M.T.J.

    2012-01-01

    This thesis describes a series of single-molecule experiments aimed at understanding the physical properties of DNA itself and the proteins that interact with it. We developed and applied sensitive techniques that allowed us to directly probe the conformation and interactions of individual DNA

  12. Single-molecule experiments in biophysics: Exploring the thermal ...

    Indian Academy of Sciences (India)

    cules contributes to our understanding of the nonequilibrium thermal behavior of small systems. Keywords. Biophysics; single-molecule experiments; fluctuation theorems. PACS Nos 05.70.Ln; 82.37.Rs; 87.10.+e; 87.15.Ya. 1. Biomolecules, molecular demons and statistical physics. Biophysics is a relatively young discipline ...

  13. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    Administrator

    Abstract. We present here a theoretical approach to compute the molecular magnetic anisotropy parameters, DM and EM for single molecule magnets in any given spin eigenstate of exchange spin Hami- ltonian. We first describe a hybrid constant MS-valence bond (VB) technique of solving spin Hamilto- nians employing ...

  14. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    We present here a theoretical approach to compute the molecular magnetic anisotropy parameters, and for single molecule magnets in any given spin eigenstate of exchange spin Hamiltonian. We first describe a hybrid constant -valence bond (VB) technique of solving spin Hamiltonians employing full spatial ...

  15. Single Molecule Study of Cellulase Hydrolysis of Crystalline Cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.-S.; Luo, Y.; Baker, J. O.; Zeng, Y.; Himmel, M. E.; Smith, S.; Ding, S.-Y.

    2009-12-01

    This report seeks to elucidate the role of cellobiohydrolase-I (CBH I) in the hydrolysis of crystalline cellulose. A single-molecule approach uses various imaging techniques to investigate the surface structure of crystalline cellulose and changes made in the structure by CBH I.

  16. Single-molecule denaturation mapping of DNA in nanofluidic channels

    DEFF Research Database (Denmark)

    Reisner, Walter; Larsen, Niels Bent; Silahtaroglu, Asli

    2010-01-01

    Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO (R)-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips...

  17. An RNA toolbox for single-molecule force spectroscopy studies

    NARCIS (Netherlands)

    Vilfan, I.D.; Kamping, W.; Van den Hout, M.; Candelli, A.; Hage, S.; Dekker, N.H.

    2007-01-01

    Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNAenzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct.

  18. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. A local view on single and coupled molecules

    NARCIS (Netherlands)

    van Dijk, E.M.H.P.; Hernando Campos, J.; Hoogenboom, Jacob; Garcia Parajo, M.F.

    2005-01-01

    The paper focuses on a novel approach to reveal ultrafast dynamics in single molecules. The main strength of the approach is towards ultrafast processes in extended multi-chromophoric molecular assemblies. Excitonically coupled systems consisting of 2 and 3 rigidly linked perylene-diimide units in a

  20. Small-molecule control of protein function through Staudinger reduction

    Science.gov (United States)

    Luo, Ji; Liu, Qingyang; Morihiro, Kunihiko; Deiters, Alexander

    2016-11-01

    Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).

  1. SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoliang Sunney [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology

    2017-03-13

    specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.

  2. Quantum-Sequencing: Fast electronic single DNA molecule sequencing

    Science.gov (United States)

    Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

    2014-03-01

    A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

  3. Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection

    OpenAIRE

    Ke, Rongqin; Nong, Rachel Yuan; Fredriksson, Simon; Landegren, Ulf; Nilsson, Mats

    2013-01-01

    Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection...

  4. Modulation of intermolecular interactions in single-molecule magnets

    Science.gov (United States)

    Heroux, Katie Jeanne

    Polynuclear manganese clusters exhibiting interesting magnetic and quantum properties have been an area of intense research since the discovery of the first single-molecule magnet (SMM) in 1993. These molecules, below their blocking temperature, function as single-domain magnetic particles which exhibit classical macroscale magnetic properties as well as quantum mechanical phenomena such as quantum tunnelling of magnetization (QTM) and quantum phase interference. The union of classical and quantum behavior in these nanomaterials makes SMMs ideal candidates for high-density information storage and quantum computing. However, environmental coupling factors (nuclear spins, phonons, neighboring molecules) must be minimized if such applications are ever to be fully realized. The focus of this work is making small structural changes in well-known manganese SMMs in order to drastically enhance the overall magnetic and quantum properties of the system. Well-isolated molecules of high crystalline quality should lead to well-defined energetic and spectral properties as well. An advantage of SMMs over bulk magnetic materials is that they can be chemically altered from a "bottom-up" approach providing a synthetic tool for tuning magnetic properties. This systematic approach is utilized in the work presented herein by incorporating bulky ligands and/or counterions to "isolate" the magnetic core of [Mn4] dicubane SMMs. Reducing intermolecular interactions in the crystal lattice (neighboring molecules, solvate molecules, dipolar interactions) is an important step toward developing viable quantum computing devices. Detailed bulk magnetic studies as well as single crystal magnetization hysteresis and high-frequency EPR studies on these sterically-isolated complexes show enhanced, and sometimes even unexpected, quantum dynamics. The importance of intra- and intermolecular interactions remains a common theme throughout this work, extending to other SMMs of various topology including

  5. Single Molecule Electrochemical Detection in Aqueous Solutions and Ionic Liquids.

    Science.gov (United States)

    Byers, Joshua C; Paulose Nadappuram, Binoy; Perry, David; McKelvey, Kim; Colburn, Alex W; Unwin, Patrick R

    2015-10-20

    Single molecule electrochemical detection (SMED) is an extremely challenging aspect of electroanalytical chemistry, requiring unconventional electrochemical cells and measurements. Here, SMED is reported using a "quad-probe" (four-channel probe) pipet cell, fabricated by depositing carbon pyrolytically into two diagonally opposite barrels of a laser-pulled quartz quadruple-barreled pipet and filling the open channels with electrolyte solution, and quasi-reference counter electrodes. A meniscus forms at the end of the probe covering the two working electrodes and is brought into contact with a substrate working electrode surface. In this way, a nanogap cell is produced whereby the two carbon electrodes in the pipet can be used to promote redox cycling of an individual molecule with the substrate. Anticorrelated currents generated at the substrate and tip electrodes, at particular distances (typically tens of nanometers), are consistent with the detection of single molecules. The low background noise realized in this droplet format opens up new opportunities in single molecule electrochemistry, including the use of ionic liquids, as well as aqueous solution, and the quantitative assessment and analysis of factors influencing redox cycling currents, due to a precisely known gap size.

  6. Investigation of polyelectrolyte desorption by single molecule force spectroscopy

    International Nuclear Information System (INIS)

    Friedsam, C; Seitz, M; Gaub, H E

    2004-01-01

    Single molecule force spectroscopy has evolved into a powerful method for the investigation of intra- and intermolecular interactions at the level of individual molecules. Many examples, including the investigation of the dynamic properties of complex biological systems as well as the properties of covalent bonds or intermolecular transitions within individual polymers, are reported in the literature. The technique has recently been extended to the systematic investigation of desorption processes of individual polyelectrolyte molecules adsorbed on generic surfaces. The stable covalent attachment of polyelectrolyte molecules to the AFM-tip provides the possibility of performing long-term measurements with the same set of molecules and therefore allows the in situ observation of the impact of environmental changes on the adsorption behaviour of individual molecules. Different types of interactions, e.g. electrostatic or hydrophobic interactions, that determine the adsorption process could be identified and characterized. The experiments provided valuable details that help to understand the nature and the properties of non-covalent interactions, which is helpful with regard to biological systems as well as for technical applications. Apart from this, desorption experiments can be utilized to characterize the properties of surfaces or polymer coatings. Therefore they represent a versatile tool that can be further developed in terms of various aspects

  7. Transition paths in single-molecule force spectroscopy.

    Science.gov (United States)

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2018-03-28

    In a typical single-molecule force spectroscopy experiment, the ends of the molecule of interest are connected by long polymer linkers to a pair of mesoscopic beads trapped in the focus of two laser beams. At constant force load, the total extension, i.e., the end-to-end distance of the molecule plus linkers, is measured as a function of time. In the simplest systems, the measured extension fluctuates about two values characteristic of folded and unfolded states, with occasional transitions between them. We have recently shown that molecular (un)folding rates can be recovered from such trajectories, with a small linker correction, as long as the characteristic time of the bead fluctuations is shorter than the residence time in the unfolded (folded) state. Here, we show that accurate measurements of the molecular transition path times require an even faster apparatus response. Transition paths, the trajectory segments in which the molecule (un)folds, are properly resolved only if the beads fluctuate more rapidly than the end-to-end distance of the molecule. Therefore, over a wide regime, the measured rates may be meaningful but not the transition path times. Analytic expressions for the measured mean transition path times are obtained for systems diffusing anisotropically on a two-dimensional free energy surface. The transition path times depend on the properties both of the molecule and of the pulling device.

  8. Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy.

    Science.gov (United States)

    Goudsmits, Joris M H; van Oijen, Antoine M; Slotboom, Dirk J

    2017-01-01

    Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data. © 2017 Elsevier Inc. All rights reserved.

  9. Spin-Spin Cross Relaxation in Single-Molecule Magnets

    Science.gov (United States)

    Wernsdorfer, W.; Bhaduri, S.; Tiron, R.; Hendrickson, D. N.; Christou, G.

    2002-10-01

    The one-body tunnel picture of single-molecule magnets (SMMs) is not always sufficient to explain the measured tunnel transitions. An improvement to the picture is proposed by including also two-body tunnel transitions such as spin-spin cross relaxation (SSCR) which are mediated by dipolar and weak superexchange interactions between molecules. A Mn4 SMM is used as a model system. At certain external fields, SSCRs lead to additional quantum resonances which show up in hysteresis loop measurements as well-defined steps. A simple model is used to explain quantitatively all observed transitions.

  10. Action spectroscopy for single-molecule reactions - Experiments and theory

    Science.gov (United States)

    Kim, Y.; Motobayashi, K.; Frederiksen, T.; Ueba, H.; Kawai, M.

    2015-05-01

    We review several representative experimental results of action spectroscopy (AS) of single molecules on metal surfaces using a scanning tunneling microscope (STM) by M. Kawai's group over last decade. The experimental procedures to observe STM-AS are described. A brief description of a low-temperature STM and experimental setup are followed by key experimental techniques of how to determine an onset bias voltage of a reaction and how to measure a current change associated with reactions and finally how to observe AS for single molecule reactions. The experimental results are presented for vibrationally mediated chemical transformation of trans-2-butene to 1.3-butadiene molecule and rotational motion of a single cis-2-butene molecule among four equivalent orientations on Pd(1 1 0). The AS obtained from the motion clearly detects more vibrational modes than inelastic electron tunneling spectroscopy with an STM. AS is demonstrated as a useful and novel single molecule vibrational spectroscopy. The AS for a lateral hopping of water dimer on Pt(1 1 1) is presented as an example of novelty. Several distinct vibrational modes are detected as the thresholds in the AS. The assignment of the vibrational modes determined from the analysis of the AS is made from a view of the adsorption geometry of hydrogen-bond donor or acceptor molecules in water dimer. A generic theory of STM-AS, i.e., a reaction rate or yield as a function of bias voltage, is presented using a single adsorbate resonance model for single molecule reactions induced by the inelastic tunneling current. Formulas for the reaction rate R (V) and Y (V) , i.e., reaction yield per electron Y (V) = eR (V) / I are derived. It provides a versatile framework to analyze any vibrationally mediated reactions of single adsorbates on metal surfaces. Numerical examples are presented to demonstrate generic features of the vibrational generation rate and Y (V) at different levels of approximations and to show how the effective

  11. Automation of a single-DNA molecule stretching device

    DEFF Research Database (Denmark)

    Sørensen, Kristian Tølbøl; Lopacinska, Joanna M.; Tommerup, Niels

    2015-01-01

    We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free...... flowing in micro- and nanofluidics emit low fluorescence and change shape, thus challenging the image analysis for machine vision. We demonstrate a set of image processing steps that increase the intrinsically low signal-to-noise ratio associated with single-molecule fluorescence microscopy. Furthermore......, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the effective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however...

  12. Influence of quantum dot labels on single molecule movement in the plasma membrane

    DEFF Research Database (Denmark)

    Clausen, Mathias P.; Lagerholm, B. Christoffer

    2011-01-01

    Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... in labeling single molecules with QDs (and other particles e.g. gold particles) are induction of cross-linking of the target molecules, which can cause activation of signaling pathways or reduced mobility, and steric hindrance as a result of the probe size. Cross-linking can be a result of the multivalent...... functionalization tag (e.g. streptavidin (sAv)) or the presence of multiple mono- or multivalent functionalization tags per QD. In this work, we have compared commercially available sAv-QDs of different sizes with custom prepared Co enzyme A (CoA)-QDs both targeting a GPI-anchored protein modified with either...

  13. Vibrationally coupled electron transport through single-molecule junctions

    Energy Technology Data Exchange (ETDEWEB)

    Haertle, Rainer

    2012-04-26

    Single-molecule junctions are among the smallest electric circuits. They consist of a molecule that is bound to a left and a right electrode. With such a molecular nanocontact, the flow of electrical currents through a single molecule can be studied and controlled. Experiments on single-molecule junctions show that a single molecule carries electrical currents that can even be in the microampere regime. Thereby, a number of transport phenomena have been observed, such as, for example, diode- or transistor-like behavior, negative differential resistance and conductance switching. An objective of this field, which is commonly referred to as molecular electronics, is to relate these transport phenomena to the properties of the molecule in the contact. To this end, theoretical model calculations are employed, which facilitate an understanding of the underlying transport processes and mechanisms. Thereby, one has to take into account that molecules are flexible structures, which respond to a change of their charge state by a profound reorganization of their geometrical structure or may even dissociate. It is thus important to understand the interrelation between the vibrational degrees of freedom of a singlemolecule junction and the electrical current flowing through the contact. In this thesis, we investigate vibrational effects in electron transport through singlemolecule junctions. For these studies, we calculate and analyze transport characteristics of both generic and first-principles based model systems of a molecular contact. To this end, we employ a master equation and a nonequilibrium Green's function approach. Both methods are suitable to describe this nonequilibrium transport problem and treat the interactions of the tunneling electrons on the molecular bridge non-perturbatively. This is particularly important with respect to the vibrational degrees of freedom, which may strongly interact with the tunneling electrons. We show in detail that the resulting

  14. Single vesicle biochips for ultra-miniaturized nanoscale fluidics and single molecule bioscience

    DEFF Research Database (Denmark)

    Christensen, Andreas Lauge; Lohr, Christina; Christensen, Sune M.

    2013-01-01

    , their fabrication via controlled self-assembly, and their characterization using fluorescence microscopy. We also highlight their applications in selected fields such as nanofluidics and single molecule bioscience. Despite their great potential for improved biocompatibility, extreme miniaturization and high...

  15. Localization microscopy: mapping cellular dynamics with single molecules.

    Science.gov (United States)

    Nelson, A J; Hess, S T

    2014-04-01

    Resolution describes the smallest details within a sample that can be recovered by a microscope lens system. For optical microscopes detecting visible light, diffraction limits the resolution to ∼200-250 nm. In contrast, localization measures the position of an isolated object using its image. Single fluorescent molecules can be localized with an uncertainty of a few tens of nanometres, and in some cases less than one nanometre. Superresolution fluorescence localization microscopy (SRFLM) images and localizes fluorescent molecules in a sample. By controlling the visibility of the fluorescent molecules with light, it is possible to cause a sparse subset of the tags to fluoresce and be spatially separated from each other. A movie is acquired with a camera, capturing images of many sets of visible fluorescent tags over a period of time. The movie is then analysed by a computer whereby all of the single molecules are independently measured, and their positions are recorded. When the coordinates of a sufficient number of molecules are collected, an image can be rendered by plotting the coordinates of the localized molecules. The spatial resolution of these rendered images can be better than 20 nm, roughly an order of magnitude better than the diffraction limited resolution. The invention of SRFLM has led to an explosion of related techniques. Through the use of specialized optics, the fluorescent signal can be split into multiple detection channels. These channels can capture additional information such as colour (emission wavelength), orientation and three-dimensional position of the detected molecules. Measurement of the colour of the detected fluorescence can allow researchers to distinguish multiple types of fluorescent tags and to study the interaction between multiple molecules of interest. Three-dimensional imaging and determination of molecular orientations offer insight into structural organization of the sample. SRFLM is compatible with living samples and

  16. Simple test system for single molecule recognition force microscopy

    International Nuclear Information System (INIS)

    Riener, Christian K.; Stroh, Cordula M.; Ebner, Andreas; Klampfl, Christian; Gall, Alex A.; Romanin, Christoph; Lyubchenko, Yuri L.; Hinterdorfer, Peter; Gruber, Hermann J.

    2003-01-01

    We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG 800 diamine was glutarylated, the mono-adduct NH 2 -PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy

  17. New Antifouling Platform Characterized by Single-Molecule Imaging

    Science.gov (United States)

    2015-01-01

    Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm2 which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm2 adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm2). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others. PMID:24503420

  18. Light-Induced Switching of Tunable Single-Molecule Junctions

    KAUST Repository

    Sendler, Torsten

    2015-04-16

    A major goal of molecular electronics is the development and implementation of devices such as single-molecular switches. Here, measurements are presented that show the controlled in situ switching of diarylethene molecules from their nonconductive to conductive state in contact to gold nanoelectrodes via controlled light irradiation. Both the conductance and the quantum yield for switching of these molecules are within a range making the molecules suitable for actual devices. The conductance of the molecular junctions in the opened and closed states is characterized and the molecular level E 0, which dominates the current transport in the closed state, and its level broadening Γ are identified. The obtained results show a clear light-induced ring forming isomerization of the single-molecule junctions. Electron withdrawing side-groups lead to a reduction of conductance, but do not influence the efficiency of the switching mechanism. Quantum chemical calculations of the light-induced switching processes correlate these observations with the fundamentally different low-lying electronic states of the opened and closed forms and their comparably small modification by electron-withdrawing substituents. This full characterization of a molecular switch operated in a molecular junction is an important step toward the development of real molecular electronics devices.

  19. Machine learning approach for single molecule localisation microscopy.

    Science.gov (United States)

    Colabrese, Silvia; Castello, Marco; Vicidomini, Giuseppe; Del Bue, Alessio

    2018-04-01

    Single molecule localisation (SML) microscopy is a fundamental tool for biological discoveries; it provides sub-diffraction spatial resolution images by detecting and localizing "all" the fluorescent molecules labeling the structure of interest. For this reason, the effective resolution of SML microscopy strictly depends on the algorithm used to detect and localize the single molecules from the series of microscopy frames. To adapt to the different imaging conditions that can occur in a SML experiment, all current localisation algorithms request, from the microscopy users, the choice of different parameters. This choice is not always easy and their wrong selection can lead to poor performance. Here we overcome this weakness with the use of machine learning. We propose a parameter-free pipeline for SML learning based on support vector machine (SVM). This strategy requires a short supervised training that consists in selecting by the user few fluorescent molecules (∼ 10-20) from the frames under analysis. The algorithm has been extensively tested on both synthetic and real acquisitions. Results are qualitatively and quantitatively consistent with the state of the art in SML microscopy and demonstrate that the introduction of machine learning can lead to a new class of algorithms competitive and conceived from the user point of view.

  20. Nonequilibrium Energetics of a Single F1-ATPase Molecule

    OpenAIRE

    Toyabe, Shoichi; Watanabe-Nakayama, Takahiro; Okamoto, Tetsuaki; Kudo, Seishi; Muneyuki, Eiro

    2010-01-01

    Molecular motors drive mechanical motions utilizing the free energy liberated from chemical reactions such as ATP hydrolysis. Although it is essential to know the efficiency of this free energy transduction, it has been a challenge due to the system's microscopic scale. Here, we evaluate the single-molecule energetics of a rotary molecular motor, F1-ATPase, by applying a recently derived nonequilibrium equality together with an electrorotation method. We show that the sum of the heat flow thr...

  1. Berry-phase blockade in single-molecule magnets

    OpenAIRE

    Gonzalez, Gabriel; Leuenberger, Michael N.

    2006-01-01

    We formulate the problem of electron transport through a single-molecule magnet (SMM) in the Coulomb blockade regime taking into account topological interference effects for the tunneling of the large spin of a SMM. The interference originates from spin Berry phases associated with different tunneling paths. We show that in the case of incoherent spin states it is essential to place the SMM between oppositely spin-polarized source and drain leads in order to detect the spin tunneling in the s...

  2. Dysprosium Acetylacetonato Single-Molecule Magnet Encapsulated in Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Ryo Nakanishi

    2016-12-01

    Full Text Available Dy single-molecule magnets (SMMs, which have several potential uses in a variety of applications, such as quantum computing, were encapsulated in multi-walled carbon nanotubes (MWCNTs by using a capillary method. Encapsulation was confirmed by using transmission electron microscopy (TEM. In alternating current magnetic measurements, the magnetic susceptibilities of the Dy acetylacetonato complexes showed clear frequency dependence even inside the MWCNTs, meaning that this hybrid can be used as magnetic materials in devices.

  3. Single-Molecule Photocurrent at a Metal-Molecule-Semiconductor Junction.

    Science.gov (United States)

    Vezzoli, Andrea; Brooke, Richard J; Higgins, Simon J; Schwarzacher, Walther; Nichols, Richard J

    2017-11-08

    We demonstrate here a new concept for a metal-molecule-semiconductor nanodevice employing Au and GaAs contacts that acts as a photodiode. Current-voltage traces for such junctions are recorded using a STM, and the "blinking" or "I(t)" method is used to record electrical behavior at the single-molecule level in the dark and under illumination, with both low and highly doped GaAs samples and with two different types of molecular bridge: nonconjugated pentanedithiol and the more conjugated 1,4-phenylene(dimethanethiol). Junctions with highly doped GaAs show poor rectification in the dark and a low photocurrent, while junctions with low doped GaAs show particularly high rectification ratios in the dark (>10 3 for a 1.5 V bias potential) and a high photocurrent in reverse bias. In low doped GaAs, the greater thickness of the depletion layer not only reduces the reverse bias leakage current, but also increases the volume that contributes to the photocurrent, an effect amplified by the point contact geometry of the junction. Furthermore, since photogenerated holes tunnel to the metal electrode assisted by the HOMO of the molecular bridge, the choice of the latter has a strong influence on both the steady state and transient metal-molecule-semiconductor photodiode response. The control of junction current via photogenerated charge carriers adds new functionality to single-molecule nanodevices.

  4. Excitonic Coupling in Linear and Trefoil Trimer Perylenediimide Molecules Probed by Single-Molecule Spectroscopy

    KAUST Repository

    Yoo, Hyejin

    2012-10-25

    Perylenediimide (PDI) molecules are promising building blocks for photophysical studies of electronic interactions within multichromophore arrays. Such PDI arrays are important materials for fabrication of molecular nanodevices such as organic light-emitting diodes, organic semiconductors, and biosensors because of their high photostability, chemical and physical inertness, electron affinity, and high tinctorial strength over the entire visible spectrum. In this work, PDIs have been organized into linear (L3) and trefoil (T3) trimer molecules and investigated by single-molecule fluorescence microscopy to probe the relationship between molecular structures and interchromophoric electronic interactions. We found a broad distribution of coupling strengths in both L3 and T3 and hence strong/weak coupling between PDI units by monitoring spectral peak shifts in single-molecule fluorescence spectra upon sequential photobleaching of each constituent chromophore. In addition, we used a wide-field defocused imaging technique to resolve heterogeneities in molecular structures of L3 and T3 embedded in a PMMA polymer matrix. A systematic comparison between the two sets of experimental results allowed us to infer the correlation between intermolecular interactions and molecular structures. Our results show control of the PDI intermolecular interactions using suitable multichromophoric structures. © 2012 American Chemical Society.

  5. Electron transfer behaviour of biological macromolecules towards the single-molecule level

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Grubb, Mikala; Hansen, Allan Glargaard

    2003-01-01

    is combined with state-of-the-art physical electrochemistry with emphasis on single-crystal, atomically planar electrode surfaces, in situ scanning tunnelling microscopy (STM) and other surface techniques. These approaches have brought bioelectrochemistry important steps forward towards the nanoscale...... and single-molecule levels.We discuss here these advances with reference to two specific redox metalloproteins, the blue single-copper protein Pseudomonas aeruginosa azurin and the single-haem protein Saccharomyces cerevisiae yeast cytochrome c, and a short oligonucleotide. Both proteins can be immobilized...... electron transfer (ET) function retained. In situ STM can also address the microscopic mechanisms for electron tunnelling through the biomolecules and offers novel notions such as coherent multi-ET between the substrate and tip via the molecular redox levels. This differs in important respects from...

  6. Single Molecule Screening of Disease DNA Without Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji-Young [Iowa State Univ., Ames, IA (United States)

    2006-01-01

    The potential of single molecule detection as an analysis tool in biological and medical fields is well recognized today. This fast evolving technique will provide fundamental sensitivity to pick up individual pathogen molecules, and therefore contribute to a more accurate diagnosis and a better chance for a complete cure. Many studies are being carried out to successfully apply this technique in real screening fields. In this dissertation, several attempts are shown that have been made to test and refine the application of the single molecule technique as a clinical screening method. A basic applicability was tested with a 100% target content sample, using electrophoretic mobility and multiple colors as identification tools. Both electrophoretic and spectral information of individual molecule were collected within a second, while the molecule travels along the flow in a capillary. Insertion of a transmission grating made the recording of the whole spectrum of a dye-stained molecule possible without adding complicated instrumental components. Collecting two kinds of information simultaneously and combining them allowed more thorough identification, up to 98.8% accuracy. Probing mRNA molecules with fluorescently labeled cDNA via hybridization was also carried out. The spectral differences among target, probe, and hybrid were interpreted in terms of dispersion distances after transmission grating, and used for the identification of each molecule. The probes were designed to have the least background when they are free, but have strong fluorescence after hybridization via fluorescence resonance energy transfer. The mRNA-cDNA hybrids were further imaged in whole blood, plasma, and saliva, to test how far a crude preparation can be tolerated. Imaging was possible with up to 50% of clear bio-matrix contents, suggesting a simple lysis and dilution would be sufficient for imaging for some cells. Real pathogen DNA of human papillomavirus (HPV) type-I6 in human genomic DNA

  7. Simulation of diffusion time of small molecules in protein crystals.

    Science.gov (United States)

    Geremia, Silvano; Campagnolo, Mara; Demitri, Nicola; Johnson, Louise N

    2006-03-01

    A simple model for evaluation of diffusion times of small molecule into protein crystals has been developed, which takes into account the physical and chemical properties both of protein crystal and the diffusing molecules. The model also includes consideration of binding and the binding affinity of a ligand to the protein. The model has been validated by simulation of experimental set-ups of several examples found in the literature. These experiments cover a wide range of situations: from small to relatively large diffusing molecules, crystals having low, medium, or high protein density, and different size. The reproduced experiments include ligand exchange in protein crystals by soaking techniques. Despite the simplifying assumptions of the model, theoretical and experimental data are in agreement with available data, with experimental diffusion times ranging from a few seconds to several hours. The method has been used successfully for planning intermediate cryotrapping experiments in maltodextrin phosphorylase crystals.

  8. Computational probing protein-protein interactions targeting small molecules.

    Science.gov (United States)

    Wang, Yong-Cui; Chen, Shi-Long; Deng, Nai-Yang; Wang, Yong

    2016-01-15

    With the booming of interactome studies, a lot of interactions can be measured in a high throughput way and large scale datasets are available. It is becoming apparent that many different types of interactions can be potential drug targets. Compared with inhibition of a single protein, inhibition of protein-protein interaction (PPI) is promising to improve the specificity with fewer adverse side-effects. Also it greatly broadens the drug target search space, which makes the drug target discovery difficult. Computational methods are highly desired to efficiently provide candidates for further experiments and hold the promise to greatly accelerate the discovery of novel drug targets. Here, we propose a machine learning method to predict PPI targets in a genomic-wide scale. Specifically, we develop a computational method, named as PrePPItar, to Predict PPIs as drug targets by uncovering the potential associations between drugs and PPIs. First, we survey the databases and manually construct a gold-standard positive dataset for drug and PPI interactions. This effort leads to a dataset with 227 associations among 63 PPIs and 113 FDA-approved drugs and allows us to build models to learn the association rules from the data. Second, we characterize drugs by profiling in chemical structure, drug ATC-code annotation, and side-effect space and represent PPI similarity by a symmetrical S-kernel based on protein amino acid sequence. Then the drugs and PPIs are correlated by Kronecker product kernel. Finally, a support vector machine (SVM), is trained to predict novel associations between drugs and PPIs. We validate our PrePPItar method on the well-established gold-standard dataset by cross-validation. We find that all chemical structure, drug ATC-code, and side-effect information are predictive for PPI target. Moreover, we can increase the PPI target prediction coverage by integrating multiple data sources. Follow-up database search and pathway analysis indicate that our new

  9. Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy

    Science.gov (United States)

    Kuzuya, Akinori; Sakai, Yusuke; Yamazaki, Takahiro; Xu, Yan; Komiyama, Makoto

    2011-01-01

    DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform. PMID:21863016

  10. Single-molecule imaging can be achieved in live obligate anaerobic bacteria

    Science.gov (United States)

    Karunatilaka, Krishanthi S.; Coupland, Ben R.; Cameron, Elizabeth A.; Martens, Eric C.; Koropatkin, Nicole K.; Biteen, Julie S.

    2013-02-01

    Single-molecule fluorescence (SMF) permits imaging with nanometer-scale resolution. This technique is particularly useful for cellular imaging as it provides a non-invasive, minimally perturbative means to examine macromolecular localization and dynamics, even in live cells. Here, we demonstrate that nanometer-scale SMF imaging can be extended to a new category of experiments: intracellular imaging of live, obligate anaerobic cells on the benchtop. We investigate the starch-utilization system (Sus) proteins in the gut symbiont Bacteroides thetaiotaomicron and discuss three different labels that we implemented to detect these proteins: fluorescent proteins, the tetracysteine-based FlAsH tag, and the enzymatic HaloTag.

  11. Single molecule transcription factor dynamics in the syncytial Drosophila embryo

    Science.gov (United States)

    Darzacq, Xavier

    During early development in the Drosophila embryo, cell fates are determined over the course of just 2 hours with exquisite spatio-temoral precision. One of the key regulators of this process is the transcription factor Bicoid which forms a concentration gradient across the long axis of the embryo. Although Bicoids' primary role is activation at the anterior, where concentrations are highest, it is also known to play a role in the posterior where there are only 100s of molecules per nucleus. Understanding how Bicoid can find its target at such low concentrations has remained intractable, largely due to the inability to perform single molecule imaging in the context of the developing embryo. Here we use lattice light sheet microscopy to overcome the technical barriers of sample thickness and auto-fluorescence to characterize the single molecule dynamics of Bicoid. We find that off-rates do not vary across the embryo and that instead the on-rates are modulated through the formation of clusters that enrich local concentration. This data is contrary to the current concentration dependent model of Bicoid function since local concentration within the nucleus is now a regulated parameter and suggests a previously unknown mechanism for regulation at extremely low concentrations.

  12. Kondo effect in single-molecule magnet transistors

    Science.gov (United States)

    Gonzalez, Gabriel; Leuenberger, Michael; Mucciolo, Eduardo

    2009-03-01

    We present a careful and thorough microscopic derivation of the anisotropic Kondo Hamiltonian for single-molecule magnet (SMM) transistors. When the molecule is strongly coupled to metallic leads, we show that by applying a transverse magnetic field it is possible to topologically induce or quench the Kondo effect in the conductance of a SMM with either an integer or a half-integer spin S>1/2. This topological Kondo effect is due to the Berry-phase interference between multiple quantum tunneling paths of the spin. We calculate the renormalized Berry-phase oscillations of the two Kondo peaks as a function of a transverse magnetic field by means of the poor man's scaling approach. We illustrate our findings with the SMM Ni4, which we propose as a possible candidate for the experimental observation of the conductance oscillations.

  13. Robust Magnetic Properties of a Sublimable Single-Molecule Magnet.

    Science.gov (United States)

    Kiefl, Evan; Mannini, Matteo; Bernot, Kevin; Yi, Xiaohui; Amato, Alex; Leviant, Tom; Magnani, Agnese; Prokscha, Thomas; Suter, Andreas; Sessoli, Roberta; Salman, Zaher

    2016-06-28

    The organization of single-molecule magnets (SMMs) on surfaces via thermal sublimation is a prerequisite for the development of future devices for spintronics exploiting the richness of properties offered by these magnetic molecules. However, a change in the SMM properties due to the interaction with specific surfaces is usually observed. Here we present a rare example of an SMM system that can be thermally sublimated on gold surfaces while maintaining its intact chemical structure and magnetic properties. Muon spin relaxation and ac susceptibility measurements are used to demonstrate that, unlike other SMMs, the magnetic properties of this system in thin films are very similar to those in the bulk, throughout the full volume of the film, including regions near the metal and vacuum interfaces. These results exhibit the robustness of chemical and magnetic properties of this complex and provide important clues for the development of nanostructures based on SMMs.

  14. Low-temperature phonoemissive tunneling rates in single molecule magnets

    Science.gov (United States)

    Liu, Yun; Garg, Anupam

    2016-03-01

    Tunneling between the two lowest energy levels of single molecule magnets with Ising type anisotropy, accompanied by the emission or absorption of phonons, is considered. Quantitatively accurate calculations of the rates for such tunneling are performed for a model Hamiltonian especially relevant to the best studied example, Fe8. Two different methods are used: high-order perturbation theory in the spin-phonon interaction and the non-Ising-symmetric parts of the spin Hamiltonian, and a novel semiclassical approach based on spin-coherent-state-path-integral instantons. The methods are found to be in good quantitative agreement with other, and consistent with previous approaches to the problem. The implications of these results for magnetization of molecular solids of these molecules are discussed briefly.

  15. Single-molecule correlated chemical probing of RNA.

    Science.gov (United States)

    Homan, Philip J; Favorov, Oleg V; Lavender, Christopher A; Kursun, Olcay; Ge, Xiyuan; Busan, Steven; Dokholyan, Nikolay V; Weeks, Kevin M

    2014-09-23

    Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA.

  16. Characterizing single-molecule FRET dynamics with probability distribution analysis.

    Science.gov (United States)

    Santoso, Yusdi; Torella, Joseph P; Kapanidis, Achillefs N

    2010-07-12

    Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single-molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two-state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis-testing models of conformational dynamics against experimental data.

  17. Influence of quantum dot labels on single molecule movement in the plasma membrane

    DEFF Research Database (Denmark)

    Clausen, Mathias P.; Lagerholm, B. Christoffer

    2011-01-01

    a biotin ligase acceptor peptide (BLAP) or an acyl carrier protein (ACP) tag, respectively. Trajectories of the differently labeled GPI-anchored molecules were recorded simultaneously in dual-color experiments at rates of ~25 -~1500 Hz. Knowing the effect of different labels is of utmost importance......Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... functionalization tag (e.g. streptavidin (sAv)) or the presence of multiple mono- or multivalent functionalization tags per QD. In this work, we have compared commercially available sAv-QDs of different sizes with custom prepared Co enzyme A (CoA)-QDs both targeting a GPI-anchored protein modified with either...

  18. Bright photoactivatable fluorophores for single-molecule imaging.

    Science.gov (United States)

    Grimm, Jonathan B; English, Brian P; Choi, Heejun; Muthusamy, Anand K; Mehl, Brian P; Dong, Peng; Brown, Timothy A; Lippincott-Schwartz, Jennifer; Liu, Zhe; Lionnet, Timothée; Lavis, Luke D

    2016-12-01

    Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

  19. Tunneling anisotropic magnetoresistance in single-molecule magnet junctions

    Science.gov (United States)

    Xie, Haiqing; Wang, Qiang; Jiao, Hujun; Liang, J.-Q.

    2012-08-01

    We theoretically investigate quantum transport through single-molecule magnet (SMM) junctions with ferromagnetic and normal-metal leads in the sequential regime. The current obtained by means of the rate-equation gives rise to the tunneling anisotropic magnetoresistance (TAMR), which varies with the angle between the magnetization direction of ferromagnetic lead and the easy axis of SMM. The angular dependence of TAMR can serve as a probe to determine experimentally the easy axis of SMM. Moreover, it is demonstrated that both the magnitude and the sign of TAMR are tunable by the bias voltage, suggesting a new spin-valve device with only one magnetic electrode in molecular spintronics.

  20. Berry-Phase Blockade in Single-Molecule Magnets

    Science.gov (United States)

    González, Gabriel; Leuenberger, Michael N.

    2007-06-01

    We formulate the problem of electron transport through a single-molecule magnet (SMM) in the Coulomb blockade regime taking into account topological interference effects for the tunneling of the large spin of a SMM. The interference originates from spin Berry phases associated with different tunneling paths. We show that, in the case of incoherent spin states, it is essential to place the SMM between oppositely spin-polarized source and drain leads in order to detect the spin tunneling in the stationary current, which exhibits topological zeros as a function of the transverse magnetic field.

  1. Nonequilibrium Energetics of Single Molecule Motor, Kinesin-1

    Science.gov (United States)

    Ariga, Takayuki; Tomishige, Michio; Mizuno, Daisuke

    2018-02-01

    Molecular motors are nonequilibrium open systems that convert chemical energy to mechanical work. Here we investigate the nonequilibrium energetics of a single molecule kinesin by measuring the motion of an attached probe particle and its response to external forces with optical tweezers. The sum of the heat dissipation estimated from the violation of the fluctuation-response relation and the output power was inconsistent with the input free energy rate, implying that internal dissipation is dominant. By using a two-state Markov model, we discuss the energy flow of the kinesin motor.

  2. Single-molecule chemical reactions on DNA origami

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther; Tørring, Thomas; Rotaru, Alexandru

    2010-01-01

    DNA nanotechnology and particularly DNA origami, in which long, single-stranded DNA molecules are folded into predetermined shapes, can be used to form complex self-assembled nanostructures. Although DNA itself has limited chemical, optical or electronic functionality, DNA nanostructures can serve...... on a DNA origami scaffold by atomic force microscopy. The high yields and chemoselectivities of successive cleavage and bond-forming reactions observed in these experiments demonstrate the feasibility of post-assembly chemical modification of DNA nanostructures and their potential use as locally...

  3. Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics.

    Science.gov (United States)

    Pirchi, Menahem; Tsukanov, Roman; Khamis, Rashid; Tomov, Toma E; Berger, Yaron; Khara, Dinesh C; Volkov, Hadas; Haran, Gilad; Nir, Eyal

    2016-12-29

    The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H 2 MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H 2 MM is based on analytical estimators for model parameters, derived using the Baum-Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H 2 MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 μs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼10 4 s -1 . The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules.

  4. Small Molecule Inhibitors of Protein Arginine Methyltransferases

    Science.gov (United States)

    Hu, Hao; Qian, Kun; Ho, Meng-Chiao; Zheng, Y. George

    2016-01-01

    Introduction Arginine methylation is an abundant posttranslational modification occurring in mammalian cells and catalyzed by protein arginine methyltransferases (PRMTs). Misregulation and aberrant expression of PRMTs are associated with various disease states, notably cancer. PRMTs are prominent therapeutic targets in drug discovery. Areas covered The authors provide an updated review of the research on the development of chemical modulators for PRMTs. Great efforts are seen in screening and designing potent and selective PRMT inhibitors, and a number of micromolar and submicromolar inhibitors have been obtained for key PRMT enzymes such as PRMT1, CARM1, and PRMT5. The authors provide a focus on their chemical structures, mechanism of action, and pharmacological activities. Pros and cons of each type of inhibitors are also discussed. Expert opinion Several key challenging issues exist in PRMT inhibitor discovery. Structural mechanisms of many PRMT inhibitors remain unclear. There lacks consistency in potency data due to divergence of assay methods and conditions. Physiologically relevant cellular assays are warranted. Substantial engagements are needed to investigate pharmacodynamics and pharmacokinetics of the new PRMT inhibitors in pertinent disease models. Discovery and evaluation of potent, isoform-selective, cell-permeable and in vivo-active PRMT modulators will continue to be an active arena of research in years ahead. PMID:26789238

  5. Magnetic Quantum Tunneling and Symmetry in Single Molecule Magnets

    Science.gov (United States)

    Kent, Andrew D.

    2003-03-01

    We have studied the symmetry of magnetic quantum tunneling (MQT) in single molecule magnets (SMMs) using a micro-Hall effect magnetometer and high field vector superconducting magnet system. In the most widely studied SMM, Mn12-acetate, an average crystal 4-fold symmetry in the magnetic response is shown to be due to local molecular environments of 2-fold symmetry that are rotated by 90 degrees with respect to one another. We attribute this to ligand disorder that leads to local rhombic distortions, a model first proposed by Cornia et al. based on x-ray diffraction data [1]. We have magnetically distilled a Mn12-acetate crystal to study a subset of these lower (2-fold) site symmetry molecules and present evidence for a spin-parity effect consistent with a local 2-fold symmetry [2]. These results highlight the importance of subtle changes in molecule environment in modulating magnetic anisotropy and MQT. [1] Cornia et al. Phys. Rev. Lett. 89, 257201 (2002) [2] E. del Barco, A. D. Kent, E. Rumberger, D. H. Hendrickson, G. Christou, submitted for publication (2002) and Europhys. Lett. 60, 768 (2002)

  6. Single molecule transistor based nanopore for the detection of nicotine

    Science.gov (United States)

    Ray, S. J.

    2014-12-01

    A nanopore based detection methodology was proposed and investigated for the detection of Nicotine. This technique uses a Single Molecular Transistor working as a nanopore operational in the Coulomb Blockade regime. When the Nicotine molecule is pulled through the nanopore area surrounded by the Source(S), Drain (D), and Gate electrodes, the charge stability diagram can detect the presence of the molecule and is unique for a specific molecular structure. Due to the weak coupling between the different electrodes which is set by the nanopore size, the molecular energy states stay almost unaffected by the electrostatic environment that can be realised from the charge stability diagram. Identification of different orientation and position of the Nicotine molecule within the nanopore area can be made from specific regions of overlap between different charge states on the stability diagram that could be used as an electronic fingerprint for detection. This method could be advantageous and useful to detect the presence of Nicotine in smoke which is usually performed using chemical chromatography techniques.

  7. Single molecule transistor based nanopore for the detection of nicotine

    Energy Technology Data Exchange (ETDEWEB)

    Ray, S. J., E-mail: ray.sjr@gmail.com [Institute of Materials Science, Technical University of Darmstadt, Alarich-Weiss-Str. 2, 64287 Darmstadt (Germany)

    2014-12-28

    A nanopore based detection methodology was proposed and investigated for the detection of Nicotine. This technique uses a Single Molecular Transistor working as a nanopore operational in the Coulomb Blockade regime. When the Nicotine molecule is pulled through the nanopore area surrounded by the Source(S), Drain (D), and Gate electrodes, the charge stability diagram can detect the presence of the molecule and is unique for a specific molecular structure. Due to the weak coupling between the different electrodes which is set by the nanopore size, the molecular energy states stay almost unaffected by the electrostatic environment that can be realised from the charge stability diagram. Identification of different orientation and position of the Nicotine molecule within the nanopore area can be made from specific regions of overlap between different charge states on the stability diagram that could be used as an electronic fingerprint for detection. This method could be advantageous and useful to detect the presence of Nicotine in smoke which is usually performed using chemical chromatography techniques.

  8. Single-Molecule Electrochemical Gating in Ionic Liquids

    DEFF Research Database (Denmark)

    Kay, Nicola J.; Higgins, Simon J.; Jeppesen, Jan O.

    2012-01-01

    The single-molecular conductance of a redox active molecular bridge has been studied in an electrochemical single-molecule transistor configuration in a room-temperature ionic liquid (RTIL). The redox active pyrrolo-tetrathiafulvalene (pTTF) moiety was attached to gold contacts at both ends through...... and decreases again as the second redox process is passed. This is described as an “off–on–off–on–off” conductance switching behavior. This molecular conductance vs electrochemical potential relation could be modeled well as a sequential two-step charge transfer process with full or partial vibrational...... relaxation. Using this view, reorganization energies of ∼1.2 eV have been estimated for both the first and second redox transitions for the pTTF bridge in the 1-butyl-3-methylimidazolium trifluoromethanesulfonate (BMIOTf) ionic liquid environment. By contrast, in aqueous environments, a much smaller...

  9. Enhancing Single Molecule Imaging in Optofluidics and Microfluidics

    Directory of Open Access Journals (Sweden)

    Andreas E. Vasdekis

    2011-08-01

    Full Text Available Microfluidics and optofluidics have revolutionized high-throughput analysis and chemical synthesis over the past decade. Single molecule imaging has witnessed similar growth, due to its capacity to reveal heterogeneities at high spatial and temporal resolutions. However, both resolution types are dependent on the signal to noise ratio (SNR of the image. In this paper, we review how the SNR can be enhanced in optofluidics and microfluidics. Starting with optofluidics, we outline integrated photonic structures that increase the signal emitted by single chromophores and minimize the excitation volume. Turning then to microfluidics, we review the compatible functionalization strategies that reduce noise stemming from non-specific interactions and architectures that minimize bleaching and blinking.

  10. Photoinduced nuclear spin conversion of methyl groups of single molecules

    International Nuclear Information System (INIS)

    Sigl, A.

    2007-01-01

    A methyl group is an outstanding quantum system due to its special symmetry properties. The threefold rotation around one of its bond is isomorphic to the group of even permutations of the remaining protons, a property which imposes severe quantum restrictions on the system, for instance a strict correlation of rotational states with nuclear spin states. The resulting long lifetimes of the rotational tunneling states of the methyl group can be exploited for applying certain high resolution optical techniques, like hole burning or single molecule spectroscopy to optically switch the methyl group from one tunneling state to another therebye changing the nuclear spin of the protons. One goal of the thesis was to perform this switching in single methyl groups. To this end the methyl group was attached to a chromophoric system, in the present case terrylene, which is well suited for single molecule spectroscopy as well as for hole burning. Experiments were performed with the bare terrylene molecule in a hexadecane lattice which served as a reference system, with alphamethyl terrylene and betamethyl terrylene, both embedded in hexadecane, too. A single molecular probe is a highly sensitive detector for dynamic lattice instabilities. Already the bare terrylene probe showed a wealth of interesting local dynamic effects of the hexadecane lattice which could be well acounted for by the assumption of two nearly degenerate sites with rather different optical and thermal properties, all of which could be determined in a quantitative fashion. As to the methylated terrylene systems, the experiments verified that for betamethyl terrylene it is indeed possible to measure rotational tunneling events in single methyl groups. However, the spectral patterns obtained was much more complicated than expected pointing to the presence of three spectroscopically different methyl groups. In order to achieve a definite assignement, molecular mechanics simulations of the terrylene probes in the

  11. Free enthalpies of replacing water molecules in protein binding pockets.

    Science.gov (United States)

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  12. High sensitivity fluorescent single particle and single molecule detection apparatus and method

    Science.gov (United States)

    Mathies, Richard A.; Peck, Konan; Stryer, Lubert

    1990-01-01

    Apparatus is described for ultrasensitive detection of single fluorescent particles down to the single fluorescent molecule limit in a fluid or on a substrate comprising means for illuminating a predetermined volume of the fluid or area of the substrate whereby to emit light including background light from the fluid and burst of photons from particles residing in the area. The photon burst is detected in real time to generate output representative signal. The signal is received and the burst of energy from the fluorescent particles is distinguished from the background energy to provide an indication of the number, location or concentration of the particles or molecules.

  13. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

    Science.gov (United States)

    Ha, Taekjip; Tinnefeld, Philip

    2012-05-01

    Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own “personalities” regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.

  14. Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines.

    Science.gov (United States)

    Babcock, Hazen P; Zhuang, Xiaowei

    2017-04-03

    The resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2-3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.

  15. Dual-Colored DNA Comb Polymers for Single Molecule Rheology

    Science.gov (United States)

    Mai, Danielle; Marciel, Amanda; Schroeder, Charles

    2014-03-01

    We report the synthesis and characterization of branched biopolymers for single molecule rheology. In our work, we utilize a hybrid enzymatic-synthetic approach to graft ``short'' DNA branches to ``long'' DNA backbones, thereby producing macromolecular DNA comb polymers. The branches and backbones are synthesized via polymerase chain reaction with chemically modified deoxyribonucleotides (dNTPs): ``short'' branches consist of Cy5-labeled dNTPs and a terminal azide group, and ``long'' backbones contain dibenzylcyclooctyne-modified (DBCO) dNTPs. In this way, we utilize strain-promoted, copper-free cycloaddition ``click'' reactions for facile grafting of azide-terminated branches at DBCO sites along backbones. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Moreover, comb polymers can be labeled with an intercalating dye (e.g., YOYO) for dual-color fluorescence imaging. We characterized these materials using gel electrophoresis, HPLC, and optical microscopy, with atomic force microscopy in progress. Overall, DNA combs are suitable for single molecule dynamics, and in this way, our work holds the potential to improve our understanding of topologically complex polymer melts and solutions.

  16. Measuring p53 Binding to Single DNA Molecules in a Nanofluidic Device

    Science.gov (United States)

    Whelsky, Amber; Gonzalez, Nicholas, Jr.; Gal, Susannah; Levy, Stephen

    2012-02-01

    Protein-DNA binding is central to several important cellular processes, for instance, the transfer of genetic information into proteins. The p53 protein plays a central role in regulating several major cell cycle pathways, in part by binding to well-characterized DNA sequences in the promoters of specific genes. Recent studies show that the most common mutation to the protein occurs in the region responsible for its binding to DNA. We have fabricated slit-like nanofluidic devices that allow us to trap and stretch single molecules of DNA containing a known recognition sequence of p53. We use fluorescent microscopy to observe the diffusion of a single p53 protein as it searches for its DNA recognition site. We measure the reaction rates of binding to selected DNA sequences as well as the one-dimensional, non-sequence specific diffusion of p53 along a stretched DNA molecule as a function of salt concentration. The mechanism of facilitated diffusion attempts to explain how proteins seem able to find their DNA target sequences much more quickly than would be expected from three-dimensional diffusion alone. We compare the observed search mechanism used by normal and mutated p53 from cancer cells to predictions based on this theory.

  17. Single-molecule conductivity of non-redox and redox molecules at pure and gold-mined Au(111)-electrode surfaces

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Chi, Qijin; Ulstrup, Jens

    media supported by comprehensive theoretical frames, have emerged as core approaches in these exciting areas. Single-molecule redox electrochemistry is rooted in two major areas. One is the preparation of well-defined (atomically planar) electrode surfaces modified by molecular monolayers (SAMs). High...... to surface-mined Au-atoms. In addition the SAMs ensure protein/enzyme immobilization gentle enough that the proteins retain electron transfer or enzyme activity in a variety of local environments. The second area is the mapping and control of the immobilized redox molecules and metalloproteins themselves...

  18. Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli

    Science.gov (United States)

    Lill, Yoriko; Kaserer, Wallace A.; Newton, Salete M.; Lill, Markus; Klebba, Phillip E.; Ritchie, Ken

    2012-08-01

    The cytoplasm of bacterial cells is filled with individual molecules and molecular complexes that rely on diffusion to bring them together for interaction. The mobility of molecules in the cytoplasm has been characterized by several techniques mainly using fluorescent probes and ensemble methods. In order to probe the microenvrionment inside the cytoplasm as viewed by an individual molecule, we have studied single green fluorescent proteins (GFPs) diffusing in the cytoplasm of Escherichia coli cells at observation at rates ranging from 60 to 1000 Hz. Over long times the diffusion shows confinement due to the geometry of the cells themselves. A simulation in model cells using the actual distribution of cell sizes found in the experiments describes accurately the experimental results as well as reveals a short time diffusion coefficient that agrees well with that determined by ensemble methods. Higher short time diffusion coefficients can be obtained by filling the simulated cell with small spheres modeling cytoplasmic molecules and, depending on the density of particles included in the modeled cytoplasm, can approach the diffusion coefficient of GFPs found in water. Thus, single-molecule tracking combined with analysis using simple simulation of Brownian motion is able to reveal the main contributors to the GFP mobility in the cytoplasm of E. coli.

  19. Reciprocal carbonyl-carbonyl interactions in small molecules and proteins.

    Science.gov (United States)

    Rahim, Abdur; Saha, Pinaki; Jha, Kunal Kumar; Sukumar, Nagamani; Sarma, Bani Kanta

    2017-07-19

    Carbonyl-carbonyl n→π* interactions where a lone pair (n) of the oxygen atom of a carbonyl group is delocalized over the π* orbital of a nearby carbonyl group have attracted a lot of attention in recent years due to their ability to affect the 3D structure of small molecules, polyesters, peptides, and proteins. In this paper, we report the discovery of a "reciprocal" carbonyl-carbonyl interaction with substantial back and forth n→π* and π→π* electron delocalization between neighboring carbonyl groups. We have carried out experimental studies, analyses of crystallographic databases and theoretical calculations to show the presence of this interaction in both small molecules and proteins. In proteins, these interactions are primarily found in polyproline II (PPII) helices. As PPII are the most abundant secondary structures in unfolded proteins, we propose that these local interactions may have implications in protein folding.Carbonyl-carbonyl π* non covalent interactions affect the structure and stability of small molecules and proteins. Here, the authors carry out experimental studies, analyses of crystallographic databases and theoretical calculations to describe an additional type of carbonyl-carbonyl interaction.

  20. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    Science.gov (United States)

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-06-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.

  1. Probing force-induced unfolding intermediates of a single staphylococcal nuclease molecule and the effect of ligand binding

    International Nuclear Information System (INIS)

    Ishii, Takaaki; Murayama, Yoshihiro; Katano, Atsuto; Maki, Kosuke; Kuwajima, Kunihiro; Sano, Masaki

    2008-01-01

    Single-molecule manipulation techniques have given experimental access to unfolding intermediates of proteins that are inaccessible in conventional experiments. A detailed characterization of the intermediates is a challenging problem that provides new possibilities for directly probing the energy landscape of proteins. We investigated single-molecule mechanical unfolding of a small globular protein, staphylococcal nuclease (SNase), using atomic force microscopy. The unfolding trajectories of the protein displayed sub-molecular and stochastic behavior with typical lengths corresponding to the size of the unfolded substructures. Our results support the view that the single protein unfolds along multiple pathways as suggested in recent theoretical studies. Moreover, we found the drastic change, caused by the ligand and inhibitor bindings, in the mechanical unfolding dynamics

  2. Hydration dynamics of protein molecules in aqueous solution: Unity ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... Dielectric dispersion and NMRD experiments have revealed that a significant fraction of water molecules in the hydration shell of various proteins do not exhibit any slowing down of dynamics. This is usually attributed to the presence of the hydrophobic residues (HBR) on the surface, although HBRs alone ...

  3. Nucleosomal arrangement affects single-molecule transcription dynamics.

    Science.gov (United States)

    Fitz, Veronika; Shin, Jaeoh; Ehrlich, Christoph; Farnung, Lucas; Cramer, Patrick; Zaburdaev, Vasily; Grill, Stephan W

    2016-10-24

    In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.

  4. Quantum turnstile operation of single-molecule magnets

    International Nuclear Information System (INIS)

    Moldoveanu, V; Dinu, I V; Tanatar, B; Moca, C P

    2015-01-01

    The time-dependent transport through single-molecule magnets coupled to magnetic or non-magnetic electrodes is studied in the framework of the generalized master equation method. We investigate the transient regime induced by the periodic switching of the source and drain contacts. If the electrodes have opposite magnetizations the quantum turnstile operation allows the stepwise writing of intermediate excited states. In turn, the transient currents provide a way to read these states. Within our approach we take into account both the uniaxial and transverse anisotropy. The latter may induce additional quantum tunneling processes which affect the efficiency of the proposed read-and-write scheme. An equally weighted mixture of molecular spin states can be prepared if one of the electrodes is ferromagnetic. (paper)

  5. Spin interactions in Graphene-Single Molecule Magnets Hybrids

    Science.gov (United States)

    Cervetti, Christian; Rettori, Angelo; Pini, Maria Gloria; Cornia, Andrea; Repollés, Aña; Luis, Fernando; Rauschenbach, Stephan; Dressel, Martin; Kern, Klaus; Burghard, Marko; Bogani, Lapo

    2014-03-01

    Graphene is a potential component of novel spintronics devices owing to its long spin diffusion length. Besides its use as spin-transport channel, graphene can be employed for the detection and manipulation of molecular spins. This requires an appropriate coupling between the sheets and the single molecular magnets (SMM). Here, we present a comprehensive characterization of graphene-Fe4 SMM hybrids. The Fe4 clusters are anchored non-covalently to the graphene following a diffusion-limited assembly and can reorganize into random networks when subjected to slightly elevated temperature. Molecules anchored on graphene sheets show unaltered static magnetic properties, whilst the quantum dynamics is profoundly modulated. Interaction with Dirac fermions becomes the dominant spin-relaxation channel, with observable effects produced by graphene phonons and reduced dipolar interactions. Coupling to graphene drives the spins over Villain's threshold, allowing the first observation of strongly-perturbative tunneling processes. Preliminary spin-transport experiments at low-temperature are further presented.

  6. Quantum Tunneling of Magnetization in Trigonal Single-Molecule Magnets

    Science.gov (United States)

    Liu, Junjie; Del Barco, Enrique; Hill, Stephen

    2012-02-01

    We perform a numerical analysis of the quantum tunneling of magnetization (QTM) that occurs in a spin S = 6 single-molecule magnet (SMM) with idealized C3 symmetry. The deconstructive points in the QTM are located by following the Berry-phase interference (BPI) oscillations. We find that the O4^3 (=12[Sz,S+^3 +S-^3 ]) operator unfreezes odd-k QTM resonances and generates three-fold patterns of BPI minima in all resonances, including k = 0! This behavior cannot be reproduced with operators that possess even rotational symmetry about the quantization axis. We find also that the k = 0 BPI minima shift away from zero longitudinal field. The wider implications of these results will be discussed in terms of the QTM behavior observed in other SMMs.

  7. A Low Spin Manganese(IV) Nitride Single Molecule Magnet.

    Science.gov (United States)

    Ding, Mei; Cutsail, George E; Aravena, Daniel; Amoza, Martín; Rouzières, Mathieu; Dechambenoit, Pierre; Losovyj, Yaroslav; Pink, Maren; Ruiz, Eliseo; Clérac, Rodolphe; Smith, Jeremy M

    2016-09-01

    Structural, spectroscopic and magnetic methods have been used to characterize the tris(carbene)borate compound PhB(MesIm) 3 Mn≡N as a four-coordinate manganese(IV) complex with a low spin ( S = 1/2) configuration. The slow relaxation of the magnetization in this complex, i.e. its single-molecule magnet (SMM) properties, is revealed under an applied dc field. Multireference quantum mechanical calculations indicate that this SMM behavior originates from an anisotropic ground doublet stabilized by spin-orbit coupling. Consistent theoretical and experiment data show that the resulting magnetization dynamics in this system is dominated by ground state quantum tunneling, while its temperature dependence is influenced by Raman relaxation.

  8. Spin thermoelectric effects in organic single-molecule devices

    Energy Technology Data Exchange (ETDEWEB)

    Wang, H.L.; Wang, M.X.; Qian, C.; Hong, X.K.; Zhang, D.B.; Liu, Y.S.; Yang, X.F., E-mail: xfyang@cslg.edu.cn

    2017-05-25

    Highlights: • A stronger spin thermoelectric performance in a polyacetylene device is observed. • For the antiferromagnetic (AFM) ordering, a transport gap is opened. Thus the thermoelectric effects are largely enhanced. - Abstract: The spin thermoelectric performance of a polyacetylene chain bridging two zigzag graphene nanoribbons (ZGNRs) is investigated based on first principles method. Two different edge spin arrangements in ZGNRs are considered. For ferromagnetic (FM) ordering, transmission eigenstates with different spin indices distributed below and above Fermi level are observed, leading directly to a strong spin thermoelectric effect in a wide temperature range. With the edge spins arranged in the antiferromagnetic (AFM) ordering, an obvious transport gap appears in the system, which greatly enhances the thermoelectric effects. The presence of a small spin splitting also induces a spin thermoelectric effect greater than the charge thermoelectric effect in certain temperature range. In general, the single-molecule junction exhibits the potential to be used for the design of perfect thermospin devices.

  9. Nanofabrication of SERS Substrates for Single/Few Molecules Detection

    KAUST Repository

    Melino, Gianluca

    2015-05-04

    Raman spectroscopy is among the most widely employed methods to investigate the properties of materials in several fields of study. Evolution in materials science allowed us to fabricate suitable substrates, at the nanoscale, capable to enhance the electromagnetic field of the signals coming from the samples which at this range turn out to be in most cases singles or a few molecules. This particular variation of the classical technique is called SERS (Surface Enanched Raman Spectroscopy). In this work, the enhancement of the electromagnetic field is obtained by manipulation of the optical properties of metals with respect to their size. By using electroless deposition (bottom up technique), gold and silver nanoparticles were deposited in nanostructured patterns obtained on silicon wafers by means of electron beam lithography (top down technique). Rhodamine 6G in aqueous solution at extremely low concentration (10-8 M) was absorbed on the resultant dimers and the collection of the Raman spectra demonstrated the high efficiency of the substrates.

  10. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    Directory of Open Access Journals (Sweden)

    Marijn T J van Loenhout

    Full Text Available The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

  11. Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

    Science.gov (United States)

    Vercoutere, Wenonah; DeGuzman, Veronica

    2003-01-01

    The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

  12. Developing DNA nanotechnology using single-molecule fluorescence.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Liber, Miran; Berger, Yaron; Nir, Eyal

    2014-06-17

    CONSPECTUS: An important effort in the DNA nanotechnology field is focused on the rational design and manufacture of molecular structures and dynamic devices made of DNA. As is the case for other technologies that deal with manipulation of matter, rational development requires high quality and informative feedback on the building blocks and final products. For DNA nanotechnology such feedback is typically provided by gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). These analytical tools provide excellent structural information; however, usually they do not provide high-resolution dynamic information. For the development of DNA-made dynamic devices such as machines, motors, robots, and computers this constitutes a major problem. Bulk-fluorescence techniques are capable of providing dynamic information, but because only ensemble averaged information is obtained, the technique may not adequately describe the dynamics in the context of complex DNA devices. The single-molecule fluorescence (SMF) technique offers a unique combination of capabilities that make it an excellent tool for guiding the development of DNA-made devices. The technique has been increasingly used in DNA nanotechnology, especially for the analysis of structure, dynamics, integrity, and operation of DNA-made devices; however, its capabilities are not yet sufficiently familiar to the community. The purpose of this Account is to demonstrate how different SMF tools can be utilized for the development of DNA devices and for structural dynamic investigation of biomolecules in general and DNA molecules in particular. Single-molecule diffusion-based Förster resonance energy transfer and alternating laser excitation (sm-FRET/ALEX) and immobilization-based total internal reflection fluorescence (TIRF) techniques are briefly described and demonstrated. To illustrate the many applications of SMF to DNA nanotechnology, examples of SMF studies of DNA hairpins and

  13. Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics

    Science.gov (United States)

    Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

    2014-01-01

    The effects of “molecular crowding” on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop–receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high–molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ −2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop–receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple “hard-sphere” treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ −5 kcal/mol free-energy stabilization at excluded volume fractions of 30%. PMID:24850865

  14. Single-molecule conductance measurements of biomolecule translocation across biomimetic nuclear pores

    Science.gov (United States)

    Dekker, Cees

    2012-02-01

    After a brief overview of our recent work on solid-state nanopores, I will present single-molecule transport data across biomimetic nanopores that contain the key regulating parts of the nuclear pore complex (NPC). The mechanism for the remarkable selectivity of NPCs has remained unclear in a large part due to difficulties in designing experiments that can probe the transport at the relevant length and time scales. Building and measuring on biomimetic NPCs provides new opportunities to address this long-standing problem. covalently tether the natively unfolded Phe-Gly rich domains (FG-domains) of human nuclear binding proteins to a solid-state nanopore (a 10-100 nm sized hole in a SiN membrane). Ionic current measurements provide a probe to monitor single molecules that traverse the pore. Translocation events are observed for transport receptors (Impβ), whereas transport of passive molecules (BSA) is found to be blocked. Interestingly, a single type of nuclear pore proteins appears already sufficient to form a selective barrier for transport. A translocation time of about 2.5 ms is measured for Impβ. This time is found to be similar for transport across Nup153 and Nup98 coated pores, although the observed ionic conductance differs between these two types of pores. We compare two simple models for the pore conductance and find, for both Nups, that the data fits best to a model with an open central channel and a condensed layer along the outer circumference of the pore. reproducing the key features of the NPC, our biomimetic approach opens the way to study a wide variety of nucleo-cytoplasmic transport processes at the single-molecule level in vitro.

  15. Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

    Science.gov (United States)

    Lukeš, Tomáš; Pospíšil, Jakub; Fliegel, Karel; Lasser, Theo; Hagen, Guy M

    2018-01-19

    Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared to organic dyes which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM data sets using a different method: super-resolution optical fluctuation imaging (SOFI). The two modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. This dataset has potential for extensive reuse. Complete raw data from SMLM experiments has typically not been published. The YFP data exhibits low signal to noise ratios, making data analysis a challenge. These data sets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3. © The Author(s) 2018. Published by Oxford University Press.

  16. Single molecule atomic force microscopy and force spectroscopy of chitosan.

    Science.gov (United States)

    Kocun, Marta; Grandbois, Michel; Cuccia, Louis A

    2011-02-01

    Atomic force microscopy (AFM) and AFM-based force spectroscopy was used to study the desorption of individual chitosan polymer chains from substrates with varying chemical composition. AFM images of chitosan adsorbed onto a flat mica substrate show elongated single strands or aggregated bundles. The aggregated state of the polymer is consistent with the high level of flexibility and mobility expected for a highly positively charged polymer strand. Conversely, the visualization of elongated strands indicated the presence of stabilizing interactions with the substrate. Surfaces with varying chemical composition (glass, self-assembled monolayer of mercaptoundecanoic acid/decanethiol and polytetrafluoroethylene (PTFE)) were probed with chitosan modified AFM tips and the corresponding desorption energies, calculated from plateau-like features, were attributed to the desorption of individual polymer strands. Desorption energies of 2.0±0.3×10(-20)J, 1.8±0.3×10(-20)J and 3.5±0.3×10(-20)J were obtained for glass, SAM of mercaptoundecanoic/dodecanethiol and PTFE, respectively. These single molecule level results can be used as a basis for investigating chitosan and chitosan-based materials for biomaterial applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Photon-Induced Magnetization Reversal in Single Molecule Magnets

    Science.gov (United States)

    Bal, Mustafa

    2005-03-01

    Single-molecule magnets (SMM) have been the subject of intensive research for more than a decade now because of their unique properties such as macroscopic quantum tunneling. Recent work in this area is focused on whether SMM are potential qubits, as proposed theoretically [1]. We use continuous millimeter wave radiation to manipulate the populations of the energy levels of a single crystal molecular magnet Fe8 [2]. When radiation is in resonance with the transitions between energy levels, the steady state magnetization exhibits dips. As expected, the magnetic field locations of these dips vary linearly with the radiation frequency. We will describe our experimental results, which provide a lower bound of 0.17 ns for transverse relaxation time. Transitions between excited states are found even though these states have negligible population at the experimental temperature. We find evidence that the sample heating is significant when the resonance condition is satisfied. Recent experiments are concentrated on the spin dynamics of Fe8 induced by pulsed radiation and results of these studies will also be presented. [1] Leuenberger, M. N. and Loss, D., Nature 410, 789 (2001). [2] M. Bal et al., Phys. Rev. B 70, 100408(R) (2004).

  18. Single-strand DNA molecule translocation through nanoelectrode gaps

    International Nuclear Information System (INIS)

    Zhao Xiongce; Payne, Christina M; Cummings, Peter T; Lee, James W

    2007-01-01

    Molecular dynamics simulations were performed to investigate the translocation of single-strand DNA through nanoscale electrode gaps under the action of a constant driving force. The application behind this theoretical study is a proposal to use nanoelectrodes as a screening gap as part of a rapid genomic sequencing device. Preliminary results from a series of simulations using various gap widths and driving forces suggest that the narrowest electrode gap that a single-strand DNA can pass is ∼1.5 nm. The minimum force required to initiate the translocation within nanoseconds is ∼0.3 nN. Simulations using DNA segments of various lengths indicate that the minimum initiation force is insensitive to the length of DNA. However, the average threading velocity of DNA varies appreciably from short to long DNA segments. We attribute such variation to the different nature of drag force experienced by the short and long DNA segments in the environment. It is found that DNA molecules deform significantly to fit in the shape of the nanogap during the translocation

  19. Computational and Experimental Insight Into Single-Molecule Piezoelectric Materials

    Science.gov (United States)

    Marvin, Christopher Wayne

    Piezoelectric materials allow for the harvesting of ambient waste energy from the environment. Producing lightweight, highly responsive materials is a challenge for this type of material, requiring polymer, foam, or bio-inspired materials. In this dissertation, I explore the origin of the piezoelectric effect in single molecules through density functional theory (DFT), analyze the piezoresponse of bio-inspired peptidic materials through the use of atomic and piezoresponse force microscopy (AFM and PFM), and develop a novel class of materials combining flexible polyurethane foams and non-piezoelectric, polar dopants. For the DFT calculations, functional group, regiochemical, and heteroatom derivatives of [6]helicene were examined for their influence on the piezoelectric response. An aza[6]helicene derivative was found to have a piezoelectric response (108 pm/V) comparable to ceramics such as lead zirconium titanate (200+ pm/V). These computed materials have the possibility to compete with current field-leading piezomaterials such as lead zirconium titanate (PZT), zinc oxide (ZnO), and polyvinylidene difluoride (PVDF) and its derivatives. The use of AFM/PFM allows for the demonstration of the piezoelectric effect of the selfassembled monolayer (SAM) peptidic systems. Through PFM, the influence that the helicity and sequence of the peptide has on the overall response of the molecule can be analyzed. Finally, development of a novel class of piezoelectrics, the foam-based materials, expands the current understanding of the qualities required for a piezoelectric material from ceramic and rigid materials to more flexible, organic materials. Through the exploration of these novel types of piezoelectric materials, new design rules and figures of merit have been developed.

  20. Force feedback effects on single molecule hopping and pulling experiments

    Science.gov (United States)

    Rico-Pasto, M.; Pastor, I.; Ritort, F.

    2018-03-01

    Single-molecule experiments with optical tweezers have become an important tool to study the properties and mechanisms of biological systems, such as cells and nucleic acids. In particular, force unzipping experiments have been used to extract the thermodynamics and kinetics of folding and unfolding reactions. In hopping experiments, a molecule executes transitions between the unfolded and folded states at a preset value of the force [constant force mode (CFM) under force feedback] or trap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extracted from the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evans model. However, hopping experiments in the CFM are known to overestimate molecular distances and folding free energies for fast transitions compared to the response time of the feedback. In contrast, kinetic rate measurements from pulling experiments have been mostly done in the PM while the CFM is seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments in a short DNA hairpin in the PM and CFM at three different temperatures (6 °C, 25 °C, and 45 °C) exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experiments in the CFM and PM perform well at 6 °C (where kinetics are slow), whereas the CFM overestimates molecular parameters at 45 °C (where kinetics are fast). In contrast, nonequilibrium pulling experiments perform well in both modes at all temperatures. This demonstrates that the same kind of feedback algorithm in the CFM leads to more reliable determination of the folding reaction parameters in irreversible pulling experiments.

  1. Calix[4]arene Based Single-Molecule Magnets

    Energy Technology Data Exchange (ETDEWEB)

    Karotsis, Georgios; Teat, Simon J.; Wernsdorfer, Wolfgang; Piligkos, Stergios; Dalgarno, Scott J.; Brechin, Euan K.

    2009-06-04

    Single-molecule magnets (SMMs) have been the subject of much interest in recent years because their molecular nature and inherent physical properties allow the crossover between classical and quantum physics to be observed. The macroscopic observation of quantum phenomena - tunneling between different spin states, quantum interference between tunnel paths - not only allows scientists to study quantum mechanical laws in great detail, but also provides model systems with which to investigate the possible implementation of spin-based solid state qubits and molecular spintronics. The isolation of small, simple SMMs is therefore an exciting prospect. To date almost all SMMs have been made via the self-assembly of 3d metal ions in the presence of bridging/chelating organic ligands. However, very recently an exciting new class of SMMs, based on 3d metal clusters (or single lanthanide ions) housed within polyoxometalates, has appeared. These types of molecule, in which the SMM is completely encapsulated within (or shrouded by) a 'protective' organic or inorganic sheath have much potential for design and manipulation: for example, for the removal of unwanted dipolar interactions, the introduction of redox activity, or to simply aid functionalization for surface grafting. Calix[4]arenes are cyclic (typically bowl-shaped) polyphenols that have been used extensively in the formation of versatile self-assembled supramolecular structures. Although many have been reported, p-{sup t}But-calix[4]arene and calix[4]arene (TBC4 and C4 respectively, Figure 1A) are frequently encountered due to (a) synthetic accessibility, and (b) vast potential for alteration at either the upper or lower rim of the macrocyclic framework. Within the field of supramolecular chemistry, TBC4 is well known for interesting polymorphic behavior and phase transformations within anti-parallel bi-layer arrays, while C4 often forms self-included trimers. The polyphenolic nature of calix[n]arenes (where

  2. Visualizing water molecules in transmembrane proteins using radiolytic labeling methods.

    Science.gov (United States)

    Orban, Tivadar; Gupta, Sayan; Palczewski, Krzysztof; Chance, Mark R

    2010-02-09

    Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of "ordered" waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.

  3. Single molecules in soft matter : a study of biomolecular conformation, heterogeneity and plasmon enhanced fluorescence

    NARCIS (Netherlands)

    Yuan, Haifeng

    2013-01-01

    We study the dynamics of single molecules and individual gold nanorods in glycerol at variable temperatures. We demonstrate temperature-cycle microscopy on FRET-labeled polyproline and double-stranded DNA molecules to access micro-second dynamics of single molecules, and reveal the influences of

  4. A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy.

    Science.gov (United States)

    Li, Hao; Yang, Haw

    2018-03-28

    This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

  5. A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy

    Science.gov (United States)

    Li, Hao; Yang, Haw

    2018-03-01

    This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

  6. Nanopore extended field-effect transistor for selective single-molecule biosensing.

    Science.gov (United States)

    Ren, Ren; Zhang, Yanjun; Nadappuram, Binoy Paulose; Akpinar, Bernice; Klenerman, David; Ivanov, Aleksandar P; Edel, Joshua B; Korchev, Yuri

    2017-09-19

    There has been a significant drive to deliver nanotechnological solutions to biosensing, yet there remains an unmet need in the development of biosensors that are affordable, integrated, fast, capable of multiplexed detection, and offer high selectivity for trace analyte detection in biological fluids. Herein, some of these challenges are addressed by designing a new class of nanoscale sensors dubbed nanopore extended field-effect transistor (nexFET) that combine the advantages of nanopore single-molecule sensing, field-effect transistors, and recognition chemistry. We report on a polypyrrole functionalized nexFET, with controllable gate voltage that can be used to switch on/off, and slow down single-molecule DNA transport through a nanopore. This strategy enables higher molecular throughput, enhanced signal-to-noise, and even heightened selectivity via functionalization with an embedded receptor. This is shown for selective sensing of an anti-insulin antibody in the presence of its IgG isotype.Efficient detection of single molecules is vital to many biosensing technologies, which require analytical platforms with high selectivity and sensitivity. Ren et al. combine a nanopore sensor and a field-effect transistor, whereby gate voltage mediates DNA and protein transport through the nanopore.

  7. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  8. Monitoring and Manipulating Motions of Single Molecules/Nanoparticles

    Science.gov (United States)

    Chen, Fang

    -nanometer pores in theory. We then experimentally studied nanoparticles diffusing on membrane filters containing 200 nm polyethyleneglycol- or C18-modified pores. Using STED microscopy, we resolved for the first time how small particles are retained by the pores. Trapping by the pore entrances rather than adsorption is responsible for the retention. Further studies on C18-modified pores showed consistency in Gibbs free energy about the retention process. In addition, in order to understand how nanoparticles interact with the surface when they are forced to be on, or very close to, the surface, we studied nanosecond rotation dynamics of gold nanorods with one end attached on the surface. We found that the nanorod motion is dominated by van der Waals interaction-induced immobilization rather Brownian rotational diffusion as previously thought. The actual rotation, during which the nanorod transits from one immobilized state to the other, slows down by 50 times. The second part of the research is the collaboration with Tour's group in Rice University. The ultimate goal is to use light to drive a motorized nanocar at ambient conditions. To fulfill this goal, we first studied the moving kinetics of adamantane-wheeled nanocars on hydroxylated and PEG-modified surfaces using single molecule fluorescence microscopy. We found that nanocars' diffusion slows down on solid surface over time, which is possibly caused by the increased hydrophobicity of the substrate surface due to the adsorbates from the air. A sticky-spots model was proposed to explain the observed slowing down. To find out whether a light-activatable motor works when it is incorporated into a nanocar, we carefully designed a series of molecules containing a regular motor, a slow motor, a nonunidirectional motor, and no motor. We found that a fast unidirectional rotating motor enhanced the diffusion of the molecule in solution upon UV-illumination. Detailed analysis suggested that the unimolecular submersible nanomachine (USN

  9. Kinetic analysis of single molecule FRET transitions without trajectories

    Science.gov (United States)

    Schrangl, Lukas; Göhring, Janett; Schütz, Gerhard J.

    2018-03-01

    Single molecule Förster resonance energy transfer (smFRET) is a popular tool to study biological systems that undergo topological transitions on the nanometer scale. smFRET experiments typically require recording of long smFRET trajectories and subsequent statistical analysis to extract parameters such as the states' lifetimes. Alternatively, analysis of probability distributions exploits the shapes of smFRET distributions at well chosen exposure times and hence works without the acquisition of time traces. Here, we describe a variant that utilizes statistical tests to compare experimental datasets with Monte Carlo simulations. For a given model, parameters are varied to cover the full realistic parameter space. As output, the method yields p-values which quantify the likelihood for each parameter setting to be consistent with the experimental data. The method provides suitable results even if the actual lifetimes differ by an order of magnitude. We also demonstrated the robustness of the method to inaccurately determine input parameters. As proof of concept, the new method was applied to the determination of transition rate constants for Holliday junctions.

  10. Fluorescence molecule counting for single-molecule studies in crowded environment of living cells without and with broken ergodicity.

    Science.gov (United States)

    Földes-Papp, Zeno; Baumann, Gerd

    2011-05-01

    We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N <1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume ΔV at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ T(m) between the measurement time T and the meaningful time T(m), which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

  11. Investigation on Single-Molecule Junctions Based on Current–Voltage Characteristics

    Directory of Open Access Journals (Sweden)

    Yuji Isshiki

    2018-02-01

    Full Text Available The relationship between the current through an electronic device and the voltage across its terminals is a current–voltage characteristic (I–V that determine basic device performance. Currently, I–V measurement on a single-molecule scale can be performed using break junction technique, where a single molecule junction can be prepared by trapping a single molecule into a nanogap between metal electrodes. The single-molecule I–Vs provide not only the device performance, but also reflect information on energy dispersion of the electronic state and the electron-molecular vibration coupling in the junction. This mini review focuses on recent representative studies on I–Vs of the single molecule junctions that cover investigation on the single-molecule diode property, the molecular vibration, and the electronic structure as a form of transmission probability, and electronic density of states, including the spin state of the single-molecule junctions. In addition, thermoelectronic measurements based on I–Vs and identification of the charged carriers (i.e., electrons or holes are presented. The analysis in the single-molecule I–Vs provides fundamental and essential information for a better understanding of the single-molecule science, and puts the single molecule junction to more practical use in molecular devices.

  12. Real-time monitoring and manipulation of single bio-molecules in free solution

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hung-Wing [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    with TIWM. FAD bound loosely in the proteins changed from the stacked conformation to the unstacked conformation upon laser excitation. FAD in free solution not only underwent conformational changes but also reacted with each other to form a dimer. Direct measurement of the single-molecule enzymatic cleavage rates of ApaI-DNA complex in the presence of various concentrations of MgCl{sub 2} solution is reported. Results suggest that there exists a distribution of ApaI conformations around the restriction site.

  13. Single-molecule transport across an individual biomimetic nuclear pore complex

    Science.gov (United States)

    Kowalczyk, Stefan W.; Kapinos, Larisa; Blosser, Timothy R.; Magalhães, Tomás; van Nies, Pauline; Lim, Roderick Y. H.; Dekker, Cees

    2011-07-01

    Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ~2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ~25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.

  14. The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

    Science.gov (United States)

    Hua, Boyang; Wang, Yanbo; Park, Seongjin; Han, Kyu Young; Singh, Digvijay; Kim, Jin H; Cheng, Wei; Ha, Taekjip

    2018-03-13

    Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

  15. Electrochemical Control of Single-Molecule Conductance by Fermi- Level Tuning and Conjugation Switching

    DEFF Research Database (Denmark)

    Baghernejad, Masoud; Zhao, Xiaotao; Ørnsø, Kristian Baruël

    2014-01-01

    Controlling charge transport through a single molecule connected to metallic electrodes remains one of the most fundamental challenges of nanoelectronics. Here we use electrochemical gating to reversibly tune the conductance of two different organic molecules, both containing anthraquinone (AQ...

  16. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

    Science.gov (United States)

    Guo, Qing; He, Yufan; Lu, H. Peter

    2015-01-01

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions. PMID:26512103

  17. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    Science.gov (United States)

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  18. Lab-on-a-chip technologies for single-molecule studies.

    Science.gov (United States)

    Zhao, Yanhui; Chen, Danqi; Yue, Hongjun; French, Jarrod B; Rufo, Joseph; Benkovic, Stephen J; Huang, Tony Jun

    2013-06-21

    Recent developments on various lab-on-a-chip techniques allow miniaturized and integrated devices to perform on-chip single-molecule studies. Fluidic-based platforms that utilize unique microscale fluidic behavior are capable of conducting single-molecule experiments with high sensitivities and throughputs, while biomolecular systems can be studied on-chip using techniques such as DNA curtains, magnetic tweezers, and solid-state nanopores. The advances of these on-chip single-molecule techniques lead to next-generation lab-on-a-chip devices, such as DNA transistors, and single-molecule real-time (SMRT) technology for rapid and low-cost whole genome DNA sequencing. In this Focus article, we will discuss some recent successes in the development of lab-on-a-chip techniques for single-molecule studies and expound our thoughts on the near future of on-chip single-molecule studies.

  19. A Single Molecule Study of Two Bacteriophage Epigenetic Switches

    Science.gov (United States)

    Wang, Haowei

    Epigenetic switches allow organisms to evolve into different states by activating/repressing different sets of genes without mutations of the underlying DNA sequence. The study of epigenetic switches is very important to understand the mechanism of human development, the origin of cancer, mental illness and fundamental processes such as gene regulation. The coliphage lambda epigenetic switch, which allows switching from lysogeny to lysis, has been studied for more than 50 years as a paradigm, and has recently received renewed attention. Atomic force microscopy (AFM) was used here to show that the lambda repressor oligomerizes on DNA, primarily as a dodecamer, to secure a DNA loop, which is the basis of the lambda switch. This study also provides support for the idea that specifically bound repressor stabilizes adjacent, non-specifically bound repressor molecules, which confers robustness to the switch. 186 is a member of a different coliphage family. One of the major differences between the two coliphage families is that lambda phages can be induced to switch from the lysogenic to the lytic state by UV radiation, but most coliphages of P2 family, to which 186 belongs, cannot. Interaction between coliphage 186 repressor and DNA is characterized by AFM and tethered particle motion (TPM). To expedite analysis of the AFM data, MatLab codes were written to automate the laborious, manual tracing procedures. The programs automatically recognize DNA segments and protein particles in an image, in order to measure the DNA length and position of bound particles as well as their height, diameter and volume. Application of these algorithms greatly improved the efficiency of AFM analysis. It was showed that 186 CI dimers form heptameric wheels, which induce DNA wrapping and different kinds of DNA looping producing various conformations of nucleoprotein complexes. Information about the dynamics of DNA wrapping and looping on 186 CI particles was also obtained by TPM.

  20. Urea transporter proteins as targets for small-molecule diuretics.

    Science.gov (United States)

    Esteva-Font, Cristina; Anderson, Marc O; Verkman, Alan S

    2015-02-01

    Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

  1. Probing static disorder in Arrhenius kinetics by single-molecule force spectroscopy.

    Science.gov (United States)

    Kuo, Tzu-Ling; Garcia-Manyes, Sergi; Li, Jingyuan; Barel, Itay; Lu, Hui; Berne, Bruce J; Urbakh, Michael; Klafter, Joseph; Fernández, Julio M

    2010-06-22

    The widely used Arrhenius equation describes the kinetics of simple two-state reactions, with the implicit assumption of a single transition state with a well-defined activation energy barrier DeltaE, as the rate-limiting step. However, it has become increasingly clear that the saddle point of the free-energy surface in most reactions is populated by ensembles of conformations, leading to nonexponential kinetics. Here we present a theory that generalizes the Arrhenius equation to include static disorder of conformational degrees of freedom as a function of an external perturbation to fully account for a diverse set of transition states. The effect of a perturbation on static disorder is best examined at the single-molecule level. Here we use force-clamp spectroscopy to study the nonexponential kinetics of single ubiquitin proteins unfolding under force. We find that the measured variance in DeltaE shows both force-dependent and independent components, where the force-dependent component scales with F(2), in excellent agreement with our theory. Our study illustrates a novel adaptation of the classical Arrhenius equation that accounts for the microscopic origins of nonexponential kinetics, which are essential in understanding the rapidly growing body of single-molecule data.

  2. Single-molecule studies of DNA transcription using atomic force microscopy

    International Nuclear Information System (INIS)

    Billingsley, Daniel J; Crampton, Neal; Thomson, Neil H; Bonass, William A; Kirkham, Jennifer

    2012-01-01

    Atomic force microscopy (AFM) can detect single biomacromolecules with a high signal-to-noise ratio on atomically flat biocompatible support surfaces, such as mica. Contrast arises from the innate forces and therefore AFM does not require imaging contrast agents, leading to sample preparation that is relatively straightforward. The ability of AFM to operate in hydrated environments, including humid air and aqueous buffers, allows structure and function of biological and biomolecular systems to be retained. These traits of the AFM are ensuring that it is being increasingly used to study deoxyribonucleic acid (DNA) structure and DNA–protein interactions down to the secondary structure level. This report focuses in particular on reviewing the applications of AFM to the study of DNA transcription in reductionist single-molecule bottom-up approaches. The technique has allowed new insights into the interactions between ribonucleic acid (RNA) polymerase to be gained and enabled quantification of some aspects of the transcription process, such as promoter location, DNA wrapping and elongation. More recently, the trend is towards studying the interactions of more than one enzyme operating on a single DNA template. These methods begin to reveal the mechanics of gene expression at the single-molecule level and will enable us to gain greater understanding of how the genome is transcribed and translated into the proteome. (topical review)

  3. Signal regulatory proteins (SIRPS) are secreted presynaptic organizing molecules.

    Science.gov (United States)

    Umemori, Hisashi; Sanes, Joshua R

    2008-12-05

    Formation of chemical synapses requires exchange of organizing signals between the synaptic partners. Using synaptic vesicle aggregation in cultured neurons as a marker of presynaptic differentiation, we purified candidate presynaptic organizers from mouse brain. A major bioactive species was the extracellular domain of signal regulatory protein alpha (SIRP-alpha), a transmembrane immunoglobulin superfamily member concentrated at synapses. The extracellular domain of SIRP-alpha is cleaved and shed in a developmentally regulated manner. The presynaptic organizing activity of SIRP-alpha is mediated in part by CD47. SIRP-alpha homologues, SIRP-beta and -gamma also have synaptic vesicle clustering activity. The effects of SIRP-alpha are distinct from those of another presynaptic organizer, FGF22: the two proteins induced vesicle clusters of different sizes, differed in their ability to promote neurite branching, and acted through different receptors and signaling pathways. SIRP family proteins may act together with other organizing molecules to pattern synapses.

  4. Explicit treatment of water molecules in data-driven protein-protein docking: the solvated HADDOCKing approach.

    NARCIS (Netherlands)

    Kastritis, P.L.; Dijk, van A.D.J.; Bonvin, A.M.

    2012-01-01

    Water molecules are active components in, literally, every biochemical event, forming hydrogen bonds, filling cavities, and mediating interactions with other (bio)molecules. Therefore, solvent drastically affects the kinetics and thermodynamics of numerous cellular events, including protein-protein

  5. Nonlinear and Nonsymmetric Single-Molecule Electronic Properties Towards Molecular Information Processing.

    Science.gov (United States)

    Tamaki, Takashi; Ogawa, Takuji

    2017-09-05

    This review highlights molecular design for nonlinear and nonsymmetric single-molecule electronic properties such as rectification, negative differential resistance, and switching, which are important components of future single-molecule information processing devices. Perspectives on integrated "molecular circuits" are also provided. Nonlinear and nonsymmetric single-molecule electronics can be designed by utilizing (1) asymmetric molecular cores, (2) asymmetric anchoring groups, (3) an asymmetric junction environment, and (4) asymmetric electrode materials. This review mainly focuses on the design of molecular cores.

  6. Identifying fluorescently labeled single molecules in image stacks using machine learning.

    Science.gov (United States)

    Rifkin, Scott A

    2011-01-01

    In the past several years, a host of new technologies have made it possible to visualize single molecules within cells and organisms (Raj et al., Nat Methods 5:877-879, 2008; Paré et al., Curr Biol 19:2037-2042, 2009; Lu and Tsourkas, Nucleic Acids Res 37:e100, 2009; Femino et al., Science 280:585-590, 1998; Rodriguez et al., Semin Cell Dev Biol 18:202-208, 2007; Betzig et al., Science 313:1642-1645, 2006; Rust et al., Nat Methods 3:793-796, 2006; Fusco et al., Curr Biol 13:161-167, 2003). Many of these are based on fluorescence, either fluorescent proteins or fluorescent dyes coupled to a molecule of interest. In many applications, the fluorescent signal is limited to a few pixels, which poses a classic signal processing problem: how can actual signal be distinguished from background noise? In this chapter, I present a MATLAB (MathWorks (2010) MATLAB. Retrieved from http://www.mathworks.com) software suite designed to work with these single-molecule visualization technologies (Rifkin (2010) spotFinding Suite. http://www.biology.ucsd.edu/labs/rifkin/software.html). It takes images or image stacks from a fluorescence microscope as input and outputs locations of the molecules. Although the software was developed for the specific application of identifying single mRNA transcripts in fixed specimens, it is more general than this and can be used and/or customized for other applications that produce localized signals embedded in a potentially noisy background. The analysis pipeline consists of the following steps: (a) create a gold-standard dataset, (b) train a machine-learning algorithm to classify image features as signal or noise depending upon user defined statistics, (c) run the machine-learning algorithm on a new dataset to identify mRNA locations, and (d) visually inspect and correct the results.

  7. Single molecule analysis of B cell receptor motion during signaling activation

    Science.gov (United States)

    Rey Suarez, Ivan; Koo, Peter; Zhou, Shu; Wheatley, Brittany; Song, Wenxia; Mochrie, Simon; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body for signs of infection in the form of antigen on the surface of antigen presenting cells. B cell receptor (BCR) binding to antigen induces a signaling cascade that leads to B cell activation and spreading. During activation, BCR form signaling microclusters that later coalesce as the cell contracts. We have studied the dynamics of BCRs on activated murine primary B cells using single particle tracking. The tracks are analyzed using perturbation expectation-maximization (pEM), a systems-level analysis, which allows identification of different short-time diffusive states from single molecule tracks. We identified four dominant diffusive states, two of which correspond to BCRs interacting with signaling molecules. For wild-type cells, the number of BCR in signaling states increases as the cell spreads and then decreases during cell contraction. In contrast, cells lacking the actin regulatory protein, N-WASP, are unable to contract and BCRs remain in the signaling states for longer times. These observations indicate that actin cytoskeleton dynamics modulate BCR diffusion and clustering. Our results provide novel information regarding the timescale of interaction between BCR and signaling molecules.

  8. Development of new microscope unit for single molecule spectroscopy under various ambient conditions

    International Nuclear Information System (INIS)

    Yamada, T; Kaji, T; Ueda, R; Otomo, A

    2013-01-01

    This paper introduces techniques we previously developed for single molecule spectroscopy and continues on to describe our studies on dipole orientation imaging of single molecules under various ambient conditions. In these studies, we successfully obtained defocused images of single perylene diimide (PDI) molecules under air, high-vacuum, and pure N 2 gas conditions by utilizing the advantages of our new microscope unit. The studies are positioned as one of the important applications of our microscope unit for single molecule spectroscopy. We expect a wide range of applications for this unit for various microscope measurements for many types of materials.

  9. Ordered array of CoPc-vacancies filled with single-molecule rotors

    Science.gov (United States)

    Xie, Zheng-Bo; Wang, Ya-Li; Tao, Min-Long; Sun, Kai; Tu, Yu-Bing; Yuan, Hong-Kuan; Wang, Jun-Zhong

    2018-05-01

    We report the highly ordered array of CoPc-vacancies and the single-molecule rotors inside the vacancies. When CoPc molecules are deposited on Cd(0001) at low-temperature, three types of molecular vacancies appeared randomly in the CoPc monolayer. Annealing the sample to higher temperature leads to the spontaneous phase separation and self-organized arrangement of the vacancies. Highly ordered arrays of two-molecule vacancies and single-molecule vacancies have been obtained. In particular, there is a rotating CoPc molecule inside each single-molecule vacancy, which constitutes the array of single-molecule rotors. These results provide a new routine to fabricate the nano-machines on a large scale.

  10. Stereoelectronic Effect-Induced Conductance Switching in Aromatic Chain Single-Molecule Junctions.

    Science.gov (United States)

    Xin, Na; Wang, Jinying; Jia, Chuancheng; Liu, Zitong; Zhang, Xisha; Yu, Chenmin; Li, Mingliang; Wang, Shuopei; Gong, Yao; Sun, Hantao; Zhang, Guanxin; Liu, Zhirong; Zhang, Guangyu; Liao, Jianhui; Zhang, Deqing; Guo, Xuefeng

    2017-02-08

    Biphenyl, as the elementary unit of organic functional materials, has been widely used in electronic and optoelectronic devices. However, over decades little has been fundamentally understood regarding how the intramolecular conformation of biphenyl dynamically affects its transport properties at the single-molecule level. Here, we establish the stereoelectronic effect of biphenyl on its electrical conductance based on the platform of graphene-molecule single-molecule junctions, where a specifically designed hexaphenyl aromatic chain molecule is covalently sandwiched between nanogapped graphene point contacts to create stable single-molecule junctions. Both theoretical and temperature-dependent experimental results consistently demonstrate that phenyl twisting in the aromatic chain molecule produces different microstates with different degrees of conjugation, thus leading to stochastic switching between high- and low-conductance states. These investigations offer new molecular design insights into building functional single-molecule electrical devices.

  11. Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy.

    Science.gov (United States)

    Anselmetti, Dario; Bartels, Frank Wilco; Becker, Anke; Decker, Björn; Eckel, Rainer; McIntosh, Matthew; Mattay, Jochen; Plattner, Patrik; Ros, Robert; Schäfer, Christian; Sewald, Norbert

    2008-02-19

    Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.

  12. Forces and conductances in a single-molecule bipyridine junction

    DEFF Research Database (Denmark)

    Stadler, Robert; Thygesen, Kristian Sommer; Jacobsen, Karsten Wedel

    2005-01-01

    Inspired by recent measurements of forces and conductances of bipyridine nanojunctions, we have performed density functional theory calculations of structure and electron transport in a bipyridine molecule attached between gold electrodes for seven different contact geometries. The calculations...

  13. Single-molecule stochastic times in a reversible bimolecular reaction.

    Science.gov (United States)

    Keller, Peter; Valleriani, Angelo

    2012-08-28

    In this work, we consider the reversible reaction between reactants of species A and B to form the product C. We consider this reaction as a prototype of many pseudobiomolecular reactions in biology, such as for instance molecular motors. We derive the exact probability density for the stochastic waiting time that a molecule of species A needs until the reaction with a molecule of species B takes place. We perform this computation taking fully into account the stochastic fluctuations in the number of molecules of species B. We show that at low numbers of participating molecules, the exact probability density differs from the exponential density derived by assuming the law of mass action. Finally, we discuss the condition of detailed balance in the exact stochastic and in the approximate treatment.

  14. Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α.

    Science.gov (United States)

    Kilic, Sinan; Felekyan, Suren; Doroshenko, Olga; Boichenko, Iuliia; Dimura, Mykola; Vardanyan, Hayk; Bryan, Louise C; Arya, Gaurav; Seidel, Claus A M; Fierz, Beat

    2018-01-16

    The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1α (HP1α), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1α residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.

  15. Topoisomerase I as a Biomarker: Detection of Activity at the Single Molecule Level

    DEFF Research Database (Denmark)

    Proszek, Joanna; Roy, Amit; Jakobsen, Ann-Katrine

    2014-01-01

    of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring......Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations...... in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement...

  16. Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

    Energy Technology Data Exchange (ETDEWEB)

    Dynan, William S.; Li, Shuyi; Mernaugh, Raymond; Wragg, Stephanie; Takeda, Yoshihiko

    2003-03-27

    Exposure to low doses of ionizing radiation is universal. The signature injury from ionizing radiation exposure is induction of DNA double-strand breaks (DSBs). The first line of defense against DSBs is direct ligation of broken DNA ends via the nonhomologous end-joining pathway. Because even a relatively high environmental exposure induces only a few DSBs per cell, our current understanding of the response to this exposure is limited by the ability to measure DSB repair events reliably in situ at a single-molecule level. To address this need, we have taken advantage of biological amplification, measuring relocalization of proteins and detection of protein phosphorylation as a surrogate for detection of broken ends themselves. We describe the use of specific antibodies to investigate the kinetics and mechanism of repair of very small numbers of DSBs in human cells by the nonhomologous end-joining pathway.

  17. Finding Order in Randomness: Single-Molecule Studies Reveal Stochastic RNA Processing | Center for Cancer Research

    Science.gov (United States)

    Producing a functional eukaryotic messenger RNA (mRNA) requires the coordinated activity of several large protein complexes to initiate transcription, elongate nascent transcripts, splice together exons, and cleave and polyadenylate the 3’ end. Kinetic competition between these various processes has been proposed to regulate mRNA maturation, but this model could lead to multiple, randomly determined, or stochastic, pathways or outcomes. Regulatory checkpoints have been suggested as a means of ensuring quality control. However, current methods have been unable to tease apart the contributions of these processes at a single gene or on a time scale that could provide mechanistic insight. To begin to investigate the kinetic relationship between transcription and splicing, Daniel Larson, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues employed a single-molecule RNA imaging approach to monitor production and processing of a human β-globin reporter gene in living cells.

  18. Probing the local environment of a single OPE3 molecule using inelastic tunneling electron spectroscopy

    NARCIS (Netherlands)

    Frisenda, R.; Perrin, M.L.; Van der Zant, H.S.J.

    2015-01-01

    We study single-molecule oligo(phenylene ethynylene)dithiol junctions by means of inelastic electron tunneling spectroscopy (IETS). The molecule is contacted with gold nano-electrodes formed with the mechanically controllable break junction technique. We record the IETS spectrum of the molecule from

  19. A polypeptide-DNA hybrid with selective linking capability applied to single molecule nano-mechanical measurements using optical tweezers.

    Directory of Open Access Journals (Sweden)

    Fatemeh Moayed

    Full Text Available Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN and the peptide StrepTag II (ST. We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST. In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV linkage. It can be used in conjunction with Neutravidin (NTV-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.

  20. Rational design of single-molecule magnets: a supramolecular approach.

    Science.gov (United States)

    Glaser, Thorsten

    2011-01-07

    Since the discovery that Mn(12)OAc acts as a single-molecule magnet (SMM), an increasing number of transition metal complexes have been demonstrated to behave as SMMs. The signature of a SMM is a slow relaxation of the magnetization at low temperatures accompanied by a magnetic hysteresis. The origin of SMM behaviour is the existence of an appreciable thermal barrier U for spin-reversal called magnetic anisotropy barrier which is related to the combination of a large total spin ground state (S(t)) and an easy-axis magnetic anisotropy. The extensive research on Mn(12)OAc and other SMMs has established more prerequisites for a rational development of new SMMs besides the high-spin ground state and the magnetic anisotropy: the symmetry should be at least C(3) to minimize the quantum tunneling of the magnetization through the anisotropy barrier but lower than cubic to avoid the cancellation of the local anisotropies upon projection onto the spin ground state. Based on these prerequisites, we have designed the ligand triplesalen which combines the phloroglucinol bridging unit for high spin ground states by the spin-polarization mechanism with a salen-like ligand environment for single-site magnetic anisotropies by a strong tetragonal ligand field. The C(3) symmetric, trinuclear complexes of the triplesalen ligand (talen(t-Bu(2)))(6-) exhibit a strong ligand folding resulting in an overall bowl-shaped molecular structure. This ligand folding preorganizes the axial coordination sites of the metal salen subunits for the complementary binding of three facial nitrogen atoms of a hexacyanometallate unit. This leads to a high driving force for the formation of heptanuclear complexes [M(t)(6)M(c)](n+) by the assembly of three molecular building blocks. Attractive van der Waals interactions of the tert-butyl phenyl units of two triplesalen trinuclear building blocks increase the driving force. In this respect, we have been able to synthesize the isostructural series [Mn(III)(6

  1. Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

    Science.gov (United States)

    Phelps, Carey; Israels, Brett; Marsh, Morgan C; von Hippel, Peter H; Marcus, Andrew H

    2016-12-29

    Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

  2. Studies of G-quadruplex DNA structures at the single molecule level

    DEFF Research Database (Denmark)

    Kragh, Sofie Louise

    2015-01-01

    Folding of G-quaduplex structures adopted by the human telomeric repeat is here studied by single molecule FRET microscopy. This method allows for the investigation of G-quadruplex structures and their conformational dynamic. Telomeres are located at the ends of our chromosomes and end in a single...... with human telomeric repeat adopt several different G-quadruplex conformations in the presence of K+ ions. G-quadruplexes inhibit telomerase activity and are therefore potential targets for anti-cancer drugs, which can be small molecule ligands capable of stabilizing G-quadruplex structures. Understanding...... range. FRET spectroscopy can be performed on an ensemble of molecules, or on the single molecule level. In single molecule FRET experiments it is possible to follow the behaviour in time for each molecule independently, allowing insight into both dynamically and statistically heterogeneous molecular...

  3. Voltage-Driven Conformational Switching with Distinct Raman Signature in a Single-Molecule Junction.

    Science.gov (United States)

    Bi, Hai; Palma, Carlos-Andres; Gong, Yuxiang; Hasch, Peter; Elbing, Mark; Mayor, Marcel; Reichert, Joachim; Barth, Johannes V

    2018-04-11

    Precisely controlling well-defined, stable single-molecule junctions represents a pillar of single-molecule electronics. Early attempts to establish computing with molecular switching arrays were partly challenged by limitations in the direct chemical characterization of metal-molecule-metal junctions. While cryogenic scanning probe studies have advanced the mechanistic understanding of current- and voltage-induced conformational switching, metal-molecule-metal conformations are still largely inferred from indirect evidence. Hence, the development of robust, chemically sensitive techniques is instrumental for advancement in the field. Here we probe the conformation of a two-state molecular switch with vibrational spectroscopy, while simultaneously operating it by means of the applied voltage. Our study emphasizes measurements of single-molecule Raman spectra in a room-temperature stable single-molecule switch presenting a signal modulation of nearly 2 orders of magnitude.

  4. Conserved linear dynamics of single-molecule Brownian motion

    Science.gov (United States)

    Serag, Maged F.; Habuchi, Satoshi

    2017-06-01

    Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

  5. Conserved linear dynamics of single-molecule Brownian motion

    KAUST Repository

    Serag, Maged F.

    2017-06-06

    Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

  6. Deformation rate controls elasticity and unfolding pathway of single tropocollagen molecules.

    Science.gov (United States)

    Gautieri, Alfonso; Buehler, Markus J; Redaelli, Alberto

    2009-04-01

    Collagen is an important structural protein in vertebrates and is responsible for the integrity of many tissues like bone, teeth, cartilage and tendon. The mechanical properties of these tissues are primarily determined by their hierarchical arrangement and the role of the collagen matrix in their structures. Here we report a series of Steered Molecular Dynamics (SMD) simulations in explicit solvent, used to elucidate the influence of the pulling rate on the Young's modulus of individual tropocollagen molecules. We stretch a collagen peptide model sequence [(Gly-Pro-Hyp)(10)](3) with pulling rates ranging from 0.01 to 100 m/s, reaching much smaller deformation rates than reported in earlier SMD studies. Our results clearly demonstrate a strong influence of the loading velocity on the observed mechanical properties. Most notably, we find that Young's modulus converges to a constant value of approximately 4 GPa tangent modulus at 8% tensile strain when the initially crimped molecule is straightened out, for pulling rates below 0.5 m/s. This enables us for the first time to predict the elastic properties of a single tropocollagen molecule at physiologically and experimentally relevant pulling rates, directly from atomistic-level calculations. At deformation rates larger than 0.5 m/s, Young's modulus increases continuously and approaches values in excess of 15 GPa for deformation rates larger than 100 m/s. The analyses of the molecular deformation mechanisms show that the tropocollagen molecule unfolds in distinctly different ways, depending on the loading rate, which explains the observation of different values of Young's modulus at different loading rates. For low pulling rates, the triple helix first uncoils completely at 10%-20% strain, then undergoes some recoiling in the opposite direction, and finally straightens for strains larger than 30%. At intermediate rates, the molecule uncoils linearly with increasing strain up to 35% strain. Finally, at higher

  7. Aligned deposition and electrical measurements on single DNA molecules

    DEFF Research Database (Denmark)

    Eidelshtein, Gennady; Kotlyar, Alexander; Hashemi, Mohtadin

    2015-01-01

    . Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin–DNA complex only the avidin moiety is strongly and irreversibly......A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)–poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg2+ ions...

  8. Handling and Sensing of Single Enzyme Molecules: From Fluorescence Detection towards Nanoscale Electrical Measurements

    DEFF Research Database (Denmark)

    Mathwig, Klaus; Chi, Qijin; Lemay, Serge G.

    2016-01-01

    advances in all-electrical single enzyme studies with a focus on recent micro- and nanofluidic tools, which offer new ways of handling and studying small numbers of molecules or even single enzyme molecules. We particularly emphasize nanofluidic devices, which enable the integration of electrochemical...

  9. Injection molded nanofluidic chips: Fabrication method and functional tests using single-molecule DNA experiments

    DEFF Research Database (Denmark)

    Utko, Pawel; Persson, Karl Fredrik; Kristensen, Anders

    2011-01-01

    We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels.......We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels....

  10. Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

    NARCIS (Netherlands)

    Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; van der Zant, Herre S. J.

    It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the

  11. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Energy Technology Data Exchange (ETDEWEB)

    Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia; Hoffbauer, Mark A.

    2017-09-12

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  12. Single-molecule analysis of DNA replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Yardimci, Hasan; Loveland, Anna B.; van Oijen, Antoine M.; Walter, Johannes C.; Mechali, Marcel

    The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the

  13. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Science.gov (United States)

    Sansinena, Jose-Maria [Los Alamos, NM; Redondo, Antonio [Los Alamos, NM; Olazabal, Virginia [Los Alamos, NM; Hoffbauer, Mark A [Los Alamos, NM; Akhadov, Elshan A [Los Alamos, NM

    2009-12-29

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  14. Electrochemical detection of single molecules using abiotic nanopores having electrically tunable dimensions

    Energy Technology Data Exchange (ETDEWEB)

    Sansinena, Jose-Maria; Redondo, Antonio; Olazabal, Virginia; Hoffbauer, Mark A.; Akhadov, Elshan A.

    2017-10-31

    A barrier structure for use in an electrochemical stochastic membrane sensor for single molecule detection. The sensor is based upon inorganic nanopores having electrically tunable dimensions. The inorganic nanopores are formed from inorganic materials and an electrically conductive polymer. Methods of making the barrier structure and sensing single molecules using the barrier structure are also described.

  15. Single molecule experimentation in biological physics: exploring the living component of soft condensed matter one molecule at a time.

    Science.gov (United States)

    Harriman, O L J; Leake, M C

    2011-12-21

    The soft matter of biological systems consists of mesoscopic length scale building blocks, composed of a variety of different types of biological molecules. Most single biological molecules are so small that 1 billion would fit on the full-stop at the end of this sentence, but collectively they carry out the vital activities in living cells whose length scale is at least three orders of magnitude greater. Typically, the number of molecules involved in any given cellular process at any one time is relatively small, and so real physiological events may often be dominated by stochastics and fluctuation behaviour at levels comparable to thermal noise, and are generally heterogeneous in nature. This challenging combination of heterogeneity and stochasticity is best investigated experimentally at the level of single molecules, as opposed to more conventional bulk ensemble-average techniques. In recent years, the use of such molecular experimental approaches has become significantly more widespread in research laboratories around the world. In this review we discuss recent experimental approaches in biological physics which can be applied to investigate the living component of soft condensed matter to a precision of a single molecule. © 2011 IOP Publishing Ltd Printed in the UK & the USA

  16. Probing single-molecule electron-hole transfer dynamics at a molecule-NiO semiconductor nanocrystalline interface.

    Science.gov (United States)

    Dhital, Bharat; Rao, Vishal Govind; Lu, H Peter

    2017-07-14

    Interfacial charge transfer dynamics in dye-sensitized NiO nanoparticles are being investigated for photocathodes in p-type dye-sensitized solar cells. In the photoreaction, after fast electron transfer from NiO to a molecule, the recombination of the hole in the nanoparticles with the electron in a reduced molecule plays an important role in the charge separation process and solar energy harvesting. Nevertheless, knowledge of the interfacial charge recombination (CR) rate and its mechanism is still limited due to the complex photoinduced electron and hole dynamics and lack of characterization of the inhomogeneity of the dynamics. Here, we report our work on probing interfacial charge recombination dynamics in Zn(ii)-5,10,15,20-tetra(3-carboxyphenyl)porphyrin (m-ZnTCPP) dye-sensitized NiO nanoparticles by correlating single-molecule fluorescence blinking dynamics with charge transfer dynamics using single-molecule photon-stamping spectroscopy. The correlated analyses of single-molecule fluorescence intensity, lifetime, and blinking reveal the intrinsic distribution and temporal fluctuation of interfacial charge transfer reactivity, which are closely related to site-specific molecular interactions and dynamics.

  17. Detection of a single enzyme molecule based on a solid-state nanopore sensor.

    Science.gov (United States)

    Tan, ShengWei; Gu, DeJian; Liu, Hang; Liu, QuanJun

    2016-04-15

    The nanopore sensor as a high-throughput and low-cost technology can detect a single molecule in a solution. In the present study, relatively large silicon nitride (Si3N4) nanopores with diameters of ∼28 and ∼88 nm were fabricated successfully using a focused Ga ion beam. We have used solid-state nanopores with various sizes to detect the single horseradish peroxidase (HRP) molecule and for the first time analyzed single HRP molecular translocation events. In addition, a real-time monitored single enzyme molecular biochemical reaction and a translocation of the product of enzyme catalysis substrates were investigated by using a Si3N4 nanopore. Our nanopore system showed a high sensitivity in detecting single enzyme molecules and a real-time monitored single enzyme molecular biochemical reaction. This method could also be significant for studying gene expression or enzyme dynamics at the single-molecule level.

  18. Protein Structure: Alignment using Mean Field Techniques and Measurement of Isolated Individual Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Blankenbecler, Richard

    2003-03-13

    Techniques originally developed in High Energy Physics have been applied to selected problems in genetics with promising results. First, this talk will briefly review the importance of protein structure from a physics point of view. Then Mean Field Techniques used in detector track fitting algorithms will be applied to the comparison of protein structures. The practical importance of such comparisons will be discussed. Second, the possibility of measuring the charge structure of ''single'' isolated molecules using the proposed SLAC Free Electron Laser will be outlined. This involves the development of an algorithm that determines the orientation of each of the many targeted identical molecules, constructs the 3-D transform from the many 2-D patterns, and finally performs an inverse fourier transform when only the magnitude of the transform is known, since the phase is not measurable.

  19. Using Synthetic Nanopores for Single-Molecule Analyses: Detecting SNPs, Trapping DNA Molecules, and the Prospects for Sequencing DNA

    Science.gov (United States)

    Dimitrov, Valentin V.

    2009-01-01

    This work focuses on studying properties of DNA molecules and DNA-protein interactions using synthetic nanopores, and it examines the prospects of sequencing DNA using synthetic nanopores. We have developed a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. There exists…

  20. Single-Photon Source for Quantum Information Based on Single Dye Molecule Fluorescence in Liquid Crystal Host

    International Nuclear Information System (INIS)

    Lukishova, S.G.; Knox, R.P.; Freivald, P.; McNamara, A.; Boyd, R.W.; Stroud, Jr. C.R.; Schmid, A.W.; Marshall, K.L.

    2006-01-01

    This paper describes a new application for liquid crystals: quantum information technology. A deterministically polarized single-photon source that efficiently produces photons exhibiting antibunching is a pivotal hardware element in absolutely secure quantum communication. Planar-aligned nematic liquid crystal hosts deterministically align the single dye molecules which produce deterministically polarized single (antibunched) photons. In addition, 1-D photonic bandgap cholesteric liquid crystals will increase single-photon source efficiency. The experiments and challenges in the observation of deterministically polarized fluorescence from single dye molecules in planar-aligned glassy nematic-liquid-crystal oligomer as well as photon antibunching in glassy cholesteric oligomer are described for the first time

  1. Molecules with multiple switching units on a Au(111) surface: self-organization and single-molecule manipulation

    Science.gov (United States)

    Mielke, Johannes; Selvanathan, Sofia; Peters, Maike; Schwarz, Jutta; Hecht, Stefan; Grill, Leonhard

    2012-10-01

    Three different molecules, each containing two azobenzene switching units, were synthesized, successfully deposited onto a Au(111) surface by sublimation and studied by scanning tunneling microscopy at low temperatures. To investigate the influence of electronic coupling between the switching units as well as to the surface, the two azo moieties were connected either via π-conjugated para-phenylene or decoupling meta-phenylene bridges, and the number of tert-butyl groups was varied in the meta-phenylene-linked derivatives. Single molecules were found to be intact after deposition as identified by their characteristic appearance in STM images. Due to their mobility on the Au(111) surface at room temperature, the molecules spontaneously formed self-organized molecular arrangements that reflected their chemical structure. While lateral displacement of the molecules was accomplished by manipulation, trans-cis isomerization processes, typical for azobenzene switches, could not be induced.

  2. BOBA FRET: bootstrap-based analysis of single-molecule FRET data.

    Directory of Open Access Journals (Sweden)

    Sebastian L B König

    Full Text Available Time-binned single-molecule Förster resonance energy transfer (smFRET experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc. are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1/IBS1 is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET, as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/.

  3. Physics of Polymers under Nanoscopic Confinement: a Single Molecule Study

    NARCIS (Netherlands)

    Keshavarz, M.

    2016-01-01

    Physicist Masoumeh Keshavarz studied the thermal motion of a fluorescently labelled, individual “reporter” polymer molecule, surrounded and entangled by a gel of similar but unlabelled polymers. Owing to their extreme length and stiffness, it is possible to follow the shape and the motion of the

  4. Computing magnetic anisotropy constants of single molecule magnets

    Indian Academy of Sciences (India)

    Administrator

    stringent requirements for a molecule to behave as a. SMM. Modelling magnetic anisotropy in these sys- tems becomes necessary for developing new SMMs with desired properties. Magnetic anisotropy of SMMs is computed by treating the anisotropy Hamiltonian as a perturbation over the Heisenberg exchange ...

  5. Single-molecule microscopy using silicone oil immersion objective lenses

    NARCIS (Netherlands)

    Hink, M.

    2012-01-01

    Microscopy techniques capable of detecting individual molecules and providing quantitative data have the potential to offer great biological insight; however, such approaches require the efficient capture of light. Here, Mark Hink explains how the use of new silicone oil immersion objective lenses

  6. Single-molecule experiments in biophysics: Exploring the thermal ...

    Indian Academy of Sciences (India)

    bath by allowing biomolecules to exchange energy with the molecules of the solvent through the breakage ... nonequilibrium process are generated from ATP consumption during the hydrolysis cycle. Often molecular ... A central notion in thermodynamics of small systems is the concept of control para- meter [5]. This is akin ...

  7. Chemical Kinetics at the Single-Molecule Level

    Science.gov (United States)

    Levitus, Marcia

    2011-01-01

    For over a century, chemists have investigated the rates of chemical reactions using experimental conditions involving huge numbers of molecules. As a consequence, the description of the kinetics of the reaction in terms of average values was good enough for all practical purposes. From the pedagogical point of view, such a description misses the…

  8. Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

    Directory of Open Access Journals (Sweden)

    Zoltán Balogh

    2015-06-01

    Full Text Available We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference.

  9. Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

    Science.gov (United States)

    Lee, Hong-Won; Kyung, Taeyoon; Yoo, Janghyun; Kim, Tackhoon; Chung, Chaeuk; Ryu, Ji Young; Lee, Hanki; Park, Kihyun; Lee, Sangkyu; Jones, Walton D.; Lim, Dae-Sik; Hyeon, Changbong; Do Heo, Won; Yoon, Tae-Young

    2013-01-01

    Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level. PMID:23422673

  10. Bianthrone in a Single-Molecule Junction: Conductance Switching with a Bistable Molecule Facilitated by Image Charge Effects

    DEFF Research Database (Denmark)

    Bjørnholm, Thomas

    2010-01-01

    Bianthrone is a sterically hindered compound that exists in the form of two nonplanar isomers. Our experimental study of single-molecule junctions with bianthrone reveals persistent switching of electric conductance at low temperatures, which can be reasonably associated with molecular isomerizat...

  11. Electronic Transport in Single Molecule Junctions: Control of the Molecule-Electrode Coupling Through Intramolecular Tunneling Barriers

    DEFF Research Database (Denmark)

    Danilov, Andrey; Kubatkin, Sergey; Kafanov, Sergey

    2008-01-01

    We report on single molecule electron transport measurements of two oligophenylenevinylene (OPV3) derivatives placed in a nanogap between gold (Au) or lead (Pb) electrodes in a field effect transistor device. Both derivatives contain thiol end groups that allow chemical binding to the electrodes...... to sequential tunneling and Coulomb blockade behavior....

  12. A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

    Directory of Open Access Journals (Sweden)

    Alexander Gust

    2014-09-01

    Full Text Available Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

  13. Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase

    Science.gov (United States)

    Wang, Quan; Serban, Andrew J.; Wachter, Rebekka M.; Moerner, W. E.

    2018-03-01

    Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ˜0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.

  14. A systematic investigation of differential effects of cell culture substrates on the extent of artifacts in single-molecule tracking.

    Directory of Open Access Journals (Sweden)

    Laura C Zanetti-Domingues

    Full Text Available Single-molecule techniques are being increasingly applied to biomedical investigation, notwithstanding the numerous challenges they pose in terms of signal-to-noise ratio issues. Non-specific binding of probes to glass substrates, in particular, can produce experimental artifacts due to spurious molecules on glass, which can be particularly deleterious in live-cell tracking experiments. In order to resolve the issue of non-specific probe binding to substrates, we performed systematic testing of a range of available surface coatings, using three different proteins, and then extended our assessment to the ability of these coatings to foster cell growth and retain non-adhesive properties. Linear PEG, a passivating agent commonly used both in immobilized-molecule single-molecule techniques and in tissue engineering, is able to both successfully repel non-specific adhesion of fluorescent probes and to foster cell growth when functionalized with appropriate adhesive peptides. Linear PEG treatment results in a significant reduction of tracking artifacts in EGFR tracking with Affibody ligands on a cell line expressing EGFR-eGFP. The findings reported herein could be beneficial to a large number of experimental situations where single-molecule or single-particle precision is required.

  15. Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

    Science.gov (United States)

    Tutkus, Marijonas; Sasnauskas, Giedrius; Rutkauskas, Danielis

    2017-12-01

    Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules. © 2017 Wiley Periodicals, Inc.

  16. Molecular dynamics simulations on aqueous two-phase systems - Single PEG-molecules in solution

    Directory of Open Access Journals (Sweden)

    Oelmeier Stefan A

    2012-08-01

    Full Text Available Abstract Background Molecular Dynamics (MD simulations are a promising tool to generate molecular understanding of processes related to the purification of proteins. Polyethylene glycols (PEG of various length are commonly used in the production and purification of proteins. The molecular mechanisms behind PEG driven precipitation, aqueous two-phase formation or the effects of PEGylation are however still poorly understood. Results In this paper, we ran MD simulations of single PEG molecules of variable length in explicitly simulated water. The resulting structures are in good agreement with experimentally determined 3D structures of PEG. The increase in surface hydrophobicity of PEG of longer chain length could be explained on an atomic scale. PEG-water interactions as well as aqueous two-phase formation in the presence of PO4 were found to be correlated to PEG surface hydrophobicity. Conclusions We were able to show that the taken MD simulation approach is capable of generating both structural data as well as molecule descriptors in agreement with experimental data. Thus, we are confident of having a good in silico representation of PEG.

  17. Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations

    KAUST Repository

    Serag, Maged F.

    2014-10-06

    Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields.

  18. Probing the local environment of a single OPE3 molecule using inelastic tunneling electron spectroscopy.

    Science.gov (United States)

    Frisenda, Riccardo; Perrin, Mickael L; van der Zant, Herre S J

    2015-01-01

    We study single-molecule oligo(phenylene ethynylene)dithiol junctions by means of inelastic electron tunneling spectroscopy (IETS). The molecule is contacted with gold nano-electrodes formed with the mechanically controllable break junction technique. We record the IETS spectrum of the molecule from direct current measurements, both as a function of time and electrode separation. We find that for fixed electrode separation the molecule switches between various configurations, which are characterized by different IETS spectra. Similar variations in the IETS signal are observed during atomic rearrangements upon stretching of the molecular junction. Using quantum chemistry calculations, we identity some of the vibrational modes which constitute a chemical fingerprint of the molecule. In addition, changes can be attributed to rearrangements of the local molecular environment, in particular at the molecule-electrode interface. This study shows the importance of taking into account the interaction with the electrodes when describing inelastic contributions to transport through single-molecule junctions.

  19. Single molecule experiments challenge the strict wave-particle dualism of light.

    Science.gov (United States)

    Greulich, Karl Otto

    2010-01-21

    Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the "single photon limit" of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. "Single photon detectors" do not meet their promise-only "photon number resolving single photon detectors" do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

  20. Single Molecule Experiments Challenge the Strict Wave-Particle Dualism of Light

    Directory of Open Access Journals (Sweden)

    Karl Otto Greulich

    2010-01-01

    Full Text Available Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the “single photon limit” of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source. “Single photon detectors” do not meet their promise―only “photon number resolving single photon detectors” do so. Particularly, the accumulation time argument, the only safe basis for the postulate of a strictly particle like photon, has so far not yet been verified.

  1. Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

    Science.gov (United States)

    Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

    2016-01-01

    The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

  2. A Synthetic Biology Project - Developing a single-molecule device for screening drug-target interactions.

    Science.gov (United States)

    Firman, Keith; Evans, Luke; Youell, James

    2012-07-16

    This review describes a European-funded project in the area of Synthetic Biology. The project seeks to demonstrate the application of engineering techniques and methodologies to the design and construction of a biosensor for detecting drug-target interactions at the single-molecule level. Production of the proteins required for the system followed the principle of previously described "bioparts" concepts (a system where a database of biological parts - promoters, genes, terminators, linking tags and cleavage sequences - is used to construct novel gene assemblies) and cassette-type assembly of gene expression systems (the concept of linking different "bioparts" to produce functional "cassettes"), but problems were quickly identified with these approaches. DNA substrates for the device were also constructed using a cassette-system. Finally, micro-engineering was used to build a magnetoresistive Magnetic Tweezer device for detection of single molecule DNA modifying enzymes (motors), while the possibility of constructing a Hall Effect version of this device was explored. The device is currently being used to study helicases from Plasmodium as potential targets for anti-malarial drugs, but we also suggest other potential uses for the device. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters.

    Science.gov (United States)

    Krebs, Arnaud R; Imanci, Dilek; Hoerner, Leslie; Gaidatzis, Dimos; Burger, Lukas; Schübeler, Dirk

    2017-08-03

    Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53.

    Science.gov (United States)

    Rajagopalan, Sridharan; Huang, Fang; Fersht, Alan R

    2011-03-01

    The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.

  5. Single-Molecule Transport at a Rectifying GaAs Contact.

    Science.gov (United States)

    Vezzoli, Andrea; Brooke, Richard J; Ferri, Nicolò; Higgins, Simon J; Schwarzacher, Walther; Nichols, Richard J

    2017-02-08

    In most single- or few-molecule devices, the contact electrodes are simple ohmic resistors. Here we describe a new type of single-molecule device in which metal and semiconductor contact electrodes impart a function, namely, current rectification, which is then modified by a molecule bridging the gap. We study junctions with the structure Au STM tip/X/n-GaAs substrate, where "X" is either a simple alkanedithiol or a conjugated unit bearing thiol/methylthiol contacts, and we detect current jumps corresponding to the attachment and detachment of single molecules. From the magnitudes of the current jumps we can deduce values for the conductance decay constant with molecule length that agree well with values determined from Au/molecule/Au junctions. The ability to impart functionality to a single-molecule device through the properties of the contacts as well as through the properties of the molecule represents a significant extension of the single-molecule electronics "tool-box".

  6. Single-molecule study of full-length NaChBac by planar lipid bilayer recording.

    Directory of Open Access Journals (Sweden)

    Andrew Jo

    Full Text Available Planar lipid bilayer device, alternatively known as BLM, is a powerful tool to study functional properties of conducting membrane proteins such as ion channels and porins. In this work, we used BLM to study the prokaryotic voltage-gated sodium channel (Nav NaChBac in a well-defined membrane environment. Navs are an essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Navs in recent years has greatly promoted the understanding of the molecular mechanism that underlies these proteins and their eukaryotic counterparts. In this work, we investigated the single-molecule conductance and ionic selectivity behavior of NaChBac. Purified NaChBac protein was first reconstituted into lipid vesicles, which is subsequently incorporated into planar lipid bilayer by fusion. At single-molecule level, we were able to observe three distinct long-lived conductance sub-states of NaChBac. Change in the membrane potential switches on the channel mainly by increasing its opening probability. In addition, we found that individual NaChBac has similar permeability for Na+, K+, and Ca2+. The single-molecule behavior of the full-length protein is essentially highly stochastic. Our results show that planar lipid bilayer device can be used to study purified ion channels at single-molecule level in an artificial environment, and such studies can reveal new protein properties that are otherwise not observable in in vivo ensemble studies.

  7. Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions

    Energy Technology Data Exchange (ETDEWEB)

    Laurence, Ted Alfred [Univ. of California, Berkeley, CA (United States)

    2002-01-01

    Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS

  8. Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions

    International Nuclear Information System (INIS)

    Laurence, Ted Alfred

    2002-01-01

    Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS

  9. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    Science.gov (United States)

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  10. Modulation and Control of Charge Transport Through Single-Molecule Junctions.

    Science.gov (United States)

    Wang, Kun; Xu, Bingqian

    2017-02-01

    The ability to modulate and control charge transport though single-molecule junction devices is crucial to achieving the ultimate goal of molecular electronics: constructing real-world-applicable electronic components from single molecules. This review aims to highlight the progress made in single-molecule electronics, emphasizing the development of molecular junction electronics in recent years. Among many techniques that attempt to wire a molecule to metallic electrodes, the single-molecule break junction (SMBJ) technique is one of the most reliable and tunable experimental platforms for achieving metal-molecule-metal configurations. It also provides great freedom to tune charge transport through the junction. Soon after the SMBJ technique was introduced, it was extensively used to measure the conductances of individual molecules; however, different conductances were obtained for the same molecule, and it proved difficult to interpret this wide distribution of experimental data. This phenomenon was later found to be mainly due to a lack of precise experimental control and advanced data analysis methods. In recent years, researchers have directed considerable effort into advancing the SMBJ technique by gaining a deeper physical understanding of charge transport through single molecules and thus enhancing its potential applicability in functional molecular-scale electronic devices, such as molecular diodes and molecular transistors. In parallel with that research, novel data analysis methods and approaches that enable the discovery of hidden yet important features in the data are being developed. This review discusses various aspects of molecular junction electronics, from the initial goal of molecular electronics, the development of experimental techniques for creating single-molecule junctions and determining single-molecule conductance, to the characterization of functional current-voltage features and the investigation of physical properties other than charge

  11. Quantum interference effects at room temperature in OPV-based single-molecule junctions

    DEFF Research Database (Denmark)

    Arroyo, Carlos R.; Frisenda, Riccardo; Moth-Poulsen, Kasper

    2013-01-01

    Interference effects on charge transport through an individual molecule can lead to a notable modulation and suppression on its conductance. In this letter, we report the observation of quantum interference effects occurring at room temperature in single-molecule junctions based on oligo(3......)-phenylenevinylene (OPV3) derivatives, in which the central benzene ring is coupled to either para- or meta-positions. Using the break-junction technique, we find that the conductance for a single meta-OPV3 molecule wired between gold electrodes is one order of magnitude smaller than that of a para-OPV3 molecule...

  12. In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

    Science.gov (United States)

    Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

    2014-10-01

    The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In

  13. Improving the representation of peptide-like inhibitor and antibiotic molecules in the Protein Data Bank.

    Science.gov (United States)

    Dutta, Shuchismita; Dimitropoulos, Dimitris; Feng, Zukang; Persikova, Irina; Sen, Sanchayita; Shao, Chenghua; Westbrook, John; Young, Jasmine; Zhuravleva, Marina A; Kleywegt, Gerard J; Berman, Helen M

    2014-06-01

    With the accumulation of a large number and variety of molecules in the Protein Data Bank (PDB) comes the need on occasion to review and improve their representation. The Worldwide PDB (wwPDB) partners have periodically updated various aspects of structural data representation to improve the integrity and consistency of the archive. The remediation effort described here was focused on improving the representation of peptide-like inhibitor and antibiotic molecules so that they can be easily identified and analyzed. Peptide-like inhibitors or antibiotics were identified in over 1000 PDB entries, systematically reviewed and represented either as peptides with polymer sequence or as single components. For the majority of the single-component molecules, their peptide-like composition was captured in a new representation, called the subcomponent sequence. A novel concept called "group" was developed for representing complex peptide-like antibiotics and inhibitors that are composed of multiple polymer and nonpolymer components. In addition, a reference dictionary was developed with detailed information about these peptide-like molecules to aid in their annotation, identification and analysis. Based on the experience gained in this remediation, guidelines, procedures, and tools were developed to annotate new depositions containing peptide-like inhibitors and antibiotics accurately and consistently. © 2013 The Authors Biopolymers Published by Wiley Periodicals, Inc.

  14. Selectivity by Small-Molecule Inhibitors of Protein Interactions Can Be Driven by Protein Surface Fluctuations

    Science.gov (United States)

    Johnson, David K.; Karanicolas, John

    2015-01-01

    Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity) can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these “distinct” pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions. PMID:25706586

  15. Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

    Science.gov (United States)

    Yan, Rui; Moon, Seonah; Kenny, Samuel J; Xu, Ke

    2018-03-20

    As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with

  16. Thermophoretic forces on DNA measured with a single-molecule spring balance

    DEFF Research Database (Denmark)

    Pedersen, Jonas Nyvold; Lüscher, Christopher James; Marie, Rodolphe

    2014-01-01

    of the thermophoretic force in a static configuration finds forces up to 130 fN. This is eleven times stronger than the force experienced by the same molecule in the same thermal gradient in bulk, where the molecule shields itself. Our stronger forces stretch the middle of the molecule up to 80% of its contour length......We stretch a single DNA molecule with thermophoretic forces and measure these forces with a spring balance: the DNA molecule itself. It is an entropic spring which we calibrate, using as a benchmark its Brownian motion in the nanochannel that contains and prestretches it. This direct measurement...

  17. Putting prions into focus: application of single molecule detection to the diagnosis of prion diseases.

    Science.gov (United States)

    Giese, A; Bieschke, J; Eigen, M; Kretzschmar, H A

    2000-01-01

    Prion diseases are characterized by the cerebral deposition of an aggregated pathological isoform of the prion protein (PrP(Sc)) which constitutes the principal component of the transmissible agent termed prion. In order to develop a highly sensitive method for the detection of PrP(Sc) aggregates in biological samples such as cerebrospinal fluid (CSF), we used a method based on Fluorescence Correlation Spectroscopy (FCS), a technique which allows detection of single fluorescently labeled molecules in solution. Within the FCS setup, fluorescent photons emitted by molecules passing an open volume element defined by the beam of an excitation laser focussed into a diffraction-limited spot are imaged confocally onto a single photon counting detector. Aggregates of PrP(Sc) could be labeled by co-aggregation of probe molecules such as monomeric recombinant PrP or PrP-specific antibodies tagged with a fluorescent dye. In addition to slow diffusion, labeled aggregates are characterized by high fluorescence intensity, which allows detection and quantification by analysis of fluorescence intensity distribution. To improve detection of rare target particles, the accessible volume element was increased by scanning for intensely fluorescent targets (SIFT). To further improve sensitivity and specificity, two different probes were used simultaneously in a two-color setup. In a diagnostic model system of CSF spiked with purified prion rods, dual-color SIFT was more sensitive than Western blot analysis. In addition, a PrP(Sc)-specific signal was also detected in a number of CSF samples derived from CJD patients but not in controls.

  18. Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin.

    Directory of Open Access Journals (Sweden)

    Ryo Iizuka

    Full Text Available Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.

  19. Force-induced chemical reactions on the metal centre in a single metalloprotein molecule.

    Science.gov (United States)

    Zheng, Peng; Arantes, Guilherme M; Field, Martin J; Li, Hongbin

    2015-06-25

    Metalloproteins play indispensable roles in biology owing to the versatile chemical reactivity of metal centres. However, studying their reactivity in many metalloproteins is challenging, as protein three-dimensional structure encloses labile metal centres, thus limiting their access to reactants and impeding direct measurements. Here we demonstrate the use of single-molecule atomic force microscopy to induce partial unfolding to expose metal centres in metalloproteins to aqueous solution, thus allowing for studying their chemical reactivity in aqueous solution for the first time. As a proof-of-principle, we demonstrate two chemical reactions for the FeS4 centre in rubredoxin: electrophilic protonation and nucleophilic ligand substitution. Our results show that protonation and ligand substitution result in mechanical destabilization of the FeS4 centre. Quantum chemical calculations corroborated experimental results and revealed detailed reaction mechanisms. We anticipate that this novel approach will provide insights into chemical reactivity of metal centres in metalloproteins under biologically more relevant conditions.

  20. Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

    Science.gov (United States)

    Xi, Zhiqun; Gao, Ying; Sirinakis, George; Guo, Honglian; Zhang, Yongli

    2012-01-01

    The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils. PMID:22451899

  1. Single-molecule electronics: Cooling individual vibrational modes by the tunneling current.

    Science.gov (United States)

    Lykkebo, Jacob; Romano, Giuseppe; Gagliardi, Alessio; Pecchia, Alessandro; Solomon, Gemma C

    2016-03-21

    Electronic devices composed of single molecules constitute the ultimate limit in the continued downscaling of electronic components. A key challenge for single-molecule electronics is to control the temperature of these junctions. Controlling heating and cooling effects in individual vibrational modes can, in principle, be utilized to increase stability of single-molecule junctions under bias, to pump energy into particular vibrational modes to perform current-induced reactions, or to increase the resolution in inelastic electron tunneling spectroscopy by controlling the life-times of phonons in a molecule by suppressing absorption and external dissipation processes. Under bias the current and the molecule exchange energy, which typically results in heating of the molecule. However, the opposite process is also possible, where energy is extracted from the molecule by the tunneling current. Designing a molecular "heat sink" where a particular vibrational mode funnels heat out of the molecule and into the leads would be very desirable. It is even possible to imagine how the vibrational energy of the other vibrational modes could be funneled into the "cooling mode," given the right molecular design. Previous efforts to understand heating and cooling mechanisms in single molecule junctions have primarily been concerned with small models, where it is unclear which molecular systems they correspond to. In this paper, our focus is on suppressing heating and obtaining current-induced cooling in certain vibrational modes. Strategies for cooling vibrational modes in single-molecule junctions are presented, together with atomistic calculations based on those strategies. Cooling and reduced heating are observed for two different cooling schemes in calculations of atomistic single-molecule junctions.

  2. Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming

    Directory of Open Access Journals (Sweden)

    Kyung Tae Lim

    2016-04-01

    Full Text Available Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps. However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic.

  3. Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming.

    Science.gov (United States)

    Lim, Kyung Tae; Lee, Seung Chan; Gao, Yimeng; Kim, Kee-Pyo; Song, Guangqi; An, Su Yeon; Adachi, Kenjiro; Jang, Yu Jin; Kim, Jonghun; Oh, Kyoung-Jin; Kwak, Tae Hwan; Hwang, Seon In; You, Jueng Soo; Ko, Kinarm; Koo, Seung-Hoi; Sharma, Amar Deep; Kim, Jong-Hoon; Hui, Lijian; Cantz, Tobias; Schöler, Hans R; Han, Dong Wook

    2016-04-26

    Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps). However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK) dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Electrostatic similarities between protein and small molecule ligands facilitate the design of protein-protein interaction inhibitors.

    Directory of Open Access Journals (Sweden)

    Arnout Voet

    Full Text Available One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs. We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs.

  5. Research Update: Molecular electronics: The single-molecule switch and transistor

    Directory of Open Access Journals (Sweden)

    Kai Sotthewes

    2014-01-01

    Full Text Available In order to design and realize single-molecule devices it is essential to have a good understanding of the properties of an individual molecule. For electronic applications, the most important property of a molecule is its conductance. Here we show how a single octanethiol molecule can be connected to macroscopic leads and how the transport properties of the molecule can be measured. Based on this knowledge we have realized two single-molecule devices: a molecular switch and a molecular transistor. The switch can be opened and closed at will by carefully adjusting the separation between the electrical contacts and the voltage drop across the contacts. This single-molecular switch operates in a broad temperature range from cryogenic temperatures all the way up to room temperature. Via mechanical gating, i.e., compressing or stretching of the octanethiol molecule, by varying the contact's interspace, we are able to systematically adjust the conductance of the electrode-octanethiol-electrode junction. This two-terminal single-molecule transistor is very robust, but the amplification factor is rather limited.

  6. Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

    Science.gov (United States)

    Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

    2018-03-27

    Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

  7. Conformational analysis of single perfluoroalkyl chains by single-molecule real-time transmission electron microscopic imaging.

    Science.gov (United States)

    Harano, Koji; Takenaga, Shinya; Okada, Satoshi; Niimi, Yoshiko; Yoshikai, Naohiko; Isobe, Hiroyuki; Suenaga, Kazu; Kataura, Hiromichi; Koshino, Masanori; Nakamura, Eiichi

    2014-01-08

    Whereas a statistical average of molecular ensembles has been the conventional source of information on molecular structures, atomic resolution movies of single organic molecules obtained by single-molecule real-time transmission electron microscopy have recently emerged as a new tool to study the time evolution of the structures of individual molecules. The present work describes a proof-of-principle study of the determination of the conformation of each C-C bond in single perfluoroalkyl fullerene molecules encapsulated in a single-walled carbon nanotube (CNT) as well as those attached to the outer surface of a carbon nanohorn (CNH). Analysis of 82 individual molecules in CNTs under a 120 kV electron beam indicated that 6% of the CF2-CF2 bonds and about 20% of the CH2-CH2 bonds in the corresponding hydrocarbon analogue are in the gauche conformation. This comparison qualitatively matches the known conformational data based on time- and molecular-average as determined for ensembles. The transmission electron microscopy images also showed that the molecules entered the CNTs predominantly in one orientation. The molecules attached on a CNH surface moved more freely and exhibited more diverse conformation than those in a CNT, suggesting the potential applicability of this method for the determination of the dynamic shape of flexible molecules and of detailed conformations. We observed little sign of any decomposition of the specimen molecules, at least up to 10(7) e·nm(-2) (electrons/nm(2)) at 120 kV acceleration voltage. Decomposition of CNHs under irradiation with a 300 kV electron beam was suppressed by cooling to 77 K, suggesting that the decomposition is a chemical process. Several lines of evidence suggest that the graphitic substrate and the attached molecules are very cold.

  8. Small-molecule stabilization of the p53 - 14-3-3 protein-protein interaction.

    Science.gov (United States)

    Doveston, Richard G; Kuusk, Ave; Andrei, Sebastian A; Leysen, Seppe; Cao, Qing; Castaldi, Maria P; Hendricks, Adam; Brunsveld, Luc; Chen, Hongming; Boyd, Helen; Ottmann, Christian

    2017-08-01

    14-3-3 proteins are positive regulators of the tumor suppressor p53, the mutation of which is implicated in many human cancers. Current strategies for targeting of p53 involve restoration of wild-type function or inhibition of the interaction with MDM2, its key negative regulator. Despite the efficacy of these strategies, the alternate approach of stabilizing the interaction of p53 with positive regulators and, thus, enhancing tumor suppressor activity, has not been explored. Here, we report the first example of small-molecule stabilization of the 14-3-3 - p53 protein-protein interaction (PPI) and demonstrate the potential of this approach as a therapeutic modality. We also observed a disconnect between biophysical and crystallographic data in the presence of a stabilizing molecule, which is unusual in 14-3-3 PPIs. © 2017 Federation of European Biochemical Societies.

  9. Protein Frustratometer 2: a tool to localize energetic frustration in protein molecules, now with electrostatics.

    Science.gov (United States)

    Parra, R Gonzalo; Schafer, Nicholas P; Radusky, Leandro G; Tsai, Min-Yeh; Guzovsky, A Brenda; Wolynes, Peter G; Ferreiro, Diego U

    2016-07-08

    The protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins' biological behavior. It compares the energy distributions of the native state with respect to structural decoys. The network of minimally frustrated interactions encompasses the folding core of the molecule. Sites of high local frustration often correlate with functional regions such as binding sites and regions involved in allosteric transitions. We present here an upgraded version of a webserver that measures local frustration. The new implementation that allows the inclusion of electrostatic energy terms, important to the interactions with nucleic acids, is significantly faster than the previous version enabling the analysis of large macromolecular complexes within a user-friendly interface. The webserver is freely available at URL: http://frustratometer.qb.fcen.uba.ar. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Helicobacter urease: Niche construction at the single molecule level

    Indian Academy of Sciences (India)

    Prakash

    each of Helicobacter and E. coli. In addition to the structural subunits; urease activity is dependent on additional proteins that either transport its co-factor, the metal ion nickel; its substrate, urea, or mediate its assembly. Their genes are organized in operons. Shift to acid pH increases transcription from some operons and ...

  11. Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton

    NARCIS (Netherlands)

    Tudor, Cicerone; te Riet, J.; Eich, C.; Harkes, R.; Smisdom, N.; Bouhuijzen Wenger, J.; Ameloot, M.; Holt, M.; Kanger, Johannes S.; Figdor, Carl; Cambi, A.; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both

  12. Proteoform-specific protein binding of small molecules in complex matrices

    Science.gov (United States)

    Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original ...

  13. Flicker Noise as a Probe of Electronic Interaction at Metal-Single Molecule Interfaces.

    Science.gov (United States)

    Adak, Olgun; Rosenthal, Ethan; Meisner, Jeffery; Andrade, Erick F; Pasupathy, Abhay N; Nuckolls, Colin; Hybertsen, Mark S; Venkataraman, Latha

    2015-06-10

    Charge transport properties of metal-molecule interfaces depend strongly on the character of molecule-electrode interactions. Although through-bond coupled systems have attracted the most attention, through-space coupling is important in molecular systems when, for example, through-bond coupling is suppressed due to quantum interference effects. To date, a probe that clearly distinguishes these two types of coupling has not yet been demonstrated. Here, we investigate the origin of flicker noise in single molecule junctions and demonstrate how the character of the molecule-electrode coupling influences the flicker noise behavior of single molecule junctions. Importantly, we find that flicker noise shows a power law dependence on conductance in all junctions studied with an exponent that can distinguish through-space and through-bond coupling. Our results provide a new and powerful tool for probing and understanding coupling at the metal-molecule interface.

  14. Probing the local environment of a single OPE3 molecule using inelastic tunneling electron spectroscopy

    Directory of Open Access Journals (Sweden)

    Riccardo Frisenda

    2015-12-01

    Full Text Available We study single-molecule oligo(phenylene ethynylenedithiol junctions by means of inelastic electron tunneling spectroscopy (IETS. The molecule is contacted with gold nano-electrodes formed with the mechanically controllable break junction technique. We record the IETS spectrum of the molecule from direct current measurements, both as a function of time and electrode separation. We find that for fixed electrode separation the molecule switches between various configurations, which are characterized by different IETS spectra. Similar variations in the IETS signal are observed during atomic rearrangements upon stretching of the molecular junction. Using quantum chemistry calculations, we identity some of the vibrational modes which constitute a chemical fingerprint of the molecule. In addition, changes can be attributed to rearrangements of the local molecular environment, in particular at the molecule–electrode interface. This study shows the importance of taking into account the interaction with the electrodes when describing inelastic contributions to transport through single-molecule junctions.

  15. The elastic theory of a single DNA molecule

    Indian Academy of Sciences (India)

    We study the elastic responses of double- (ds) and single-stranded (ss) DNA at external force fields. A double-strand-polymer elastic model is constructed and solved by path integral methods and Monte Carlo simulations to understand the entropic elasticity, cooperative extensibility, and supercoiling property of dsDNA.

  16. The elastic theory of a single DNA molecule

    Indian Academy of Sciences (India)

    Abstract. We study the elastic responses of double- (ds) and single-stranded (ss) DNA at exter- nal force fields. A double-strand-polymer elastic model is constructed and solved by path integral methods and Monte Carlo simulations to understand the entropic elasticity, cooperative extensibil- ity, and supercoiling property of ...

  17. New tools to study biophysical properties of single molecules and single cells

    Directory of Open Access Journals (Sweden)

    Márcio S. Rocha

    2007-03-01

    Full Text Available We present a review on two new tools to study biophysical properties of single molecules and single cells. A laser incident through a high numerical aperture microscope objective can trap small dielectric particles near the focus. This arrangement is named optical tweezers. This technique has the advantage to permit manipulation of a single individual object. We use optical tweezers to measure the entropic elasticity of a single DNA molecule and its interaction with the drug Psoralen. Optical tweezers are also used to hold a kidney cell MDCK away from the substrate to allow precise volume measurements of this single cell during an osmotic shock. This procedure allows us to obtain information about membrane water permeability and regulatory volume increase. Defocusing microscopy is a recent technique invented in our laboratory, which allows the observation of transparent objects, by simply defocusing the microscope in a controlled way. Our physical model of a defocused microscope shows that the image contrast observed in this case is proportional to the defocus distance and to the curvature of the transparent object. Defocusing microscopy is very useful to study motility and mechanical properties of cells. We show here the application of defocusing microscopy to measurements of macrophage surface fluctuations and their influence on phagocytosis.Apresentamos uma revisão de duas novas técnicas para estudar propriedades biofísicas de moléculas únicas e células únicas. Um laser incidindo em uma objetiva de microscópio de grande abertura numérica é capaz de aprisionar pequenas partículas dielétricas na região próxima ao foco. Este aparato é chamado de pinça óptica. Esta técnica tem a grande vantagem de permitir a manipulação de um objeto individual. Usamos a pinça óptica para medir a elasticidade entrópica de uma molécula única de DNA em sua interação com o fármaco Psoralen. A pinça óptica também é usada para segurar

  18. Optical and Electrical Detection of Single Molecule Translocation through Carbon Nanotubes

    Science.gov (United States)

    Song, Weisi; Pang, Pei; He, Jin; Lindsay, Stuart

    2013-01-01

    Ion current through a single-walled carbon nanotube (SWCNT) was monitored at the same time as fluorescence was recorded from charged dye molecules translocating through the SWCNT. Fluorescence bursts generally follow ion-current peaks with a delay time consistent with diffusion from the end of the SWCNT to the fluorescence collection point. The fluorescence amplitude distribution of the bursts is consistent with single molecule signals. Thus each peak in the ion current flowing through the SWCNT is associated with the translocation of a single molecule. Ion current peaks (as opposed to blockades) were produced by both positively (Rhodamine 6G) and negatively (Alexa 546) charged molecules, showing that the charge filtering responsible for the current bursts is caused by the molecules themselves. PMID:23248975

  19. Estimating single molecule conductance from spontaneous evolution of a molecular contact

    Science.gov (United States)

    Gil, M.; Malinowski, T.; Iazykov, M.; Klein, H. R.

    2018-03-01

    We present an original method to estimate the conductivity of a single molecule anchored to nanometric-sized metallic electrodes, using a Mechanically Controlled Break Junction operated at room temperature in the liquid. We record the conductance through the metal/molecules/metal nanocontact while keeping the metallic electrodes at a fixed distance. Taking advantage of thermal diffusion and electromigration, we let the contact naturally explore the more stable configurations around a chosen conductance value. The conductance of a single molecule is estimated from a statistical analysis of raw conductance and conductance standard deviation data for molecular contacts containing up to 14 molecules. The single molecule conductance values are interpreted as time-averaged conductance of an ensemble of conformers at thermal equilibrium.

  20. Studying Chemical Reactions, One Bond at a Time, with Single Molecule AFM Techniques

    Science.gov (United States)

    Fernandez, Julio M.

    2008-03-01

    The mechanisms by which mechanical forces regulate the kinetics of a chemical reaction are unknown. In my lecture I will demonstrate how we use single molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bond via the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is of common occurrence in mechanically stressed proteins. While reduction is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction, the role of a mechanical force in modulating this chemical reaction is unknown. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by dithiothreitol (DTT). We find that while the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300 pN range. This result predicts that the disulfide bond lengthens by 0.34 å at the transition state of the thiol/disulfide exchange reaction. In addition to DTT, we also study the reduction of the engineered disulfide bond by the E. coli enzyme thioredoxin (Trx). Thioredoxins are enzymes that catalyze disulfide bond reduction in all organisms. As before, we apply a mechanical force in the range of 25-450 pN to the engineered disulfide bond substrate and monitor the reduction of these bonds by individual enzymes. In sharp contrast with the data obtained with DTT, we now observe two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulfide bond, causing a shortening of the substrate polypeptide by 0.76±0.07 å, and the second elongating the substrate disulfide bond by 0.21±0.01 å. These results support the view that the Trx active site regulates the geometry of the participating sulfur atoms, with sub-ångström precision, in order to achieve efficient catalysis. Single molecule

  1. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

    KAUST Repository

    Rashid, Fahad

    2017-02-23

    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. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

  2. Dipolar molecules inside C-70: an electric field-driven room-temperature single-molecule switch

    Czech Academy of Sciences Publication Activity Database

    Foroutan-Nejad, C.; Andrushchenko, Valery; Straka, Michal

    2016-01-01

    Roč. 18, č. 48 (2016), s. 32673-32677 ISSN 1463-9076 R&D Projects: GA ČR(CZ) GA14-03564S Institutional support: RVO:61388963 Keywords : room-temperature single-molecule switch * electric field * endohedral fullerene * density functional calculations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.123, year: 2016 http://pubs.rsc.org/en/content/articlepdf/2016/cp/c6cp06986j

  3. Detection of kinetic change points in piece-wise linear single molecule motion

    Science.gov (United States)

    Hill, Flynn R.; van Oijen, Antoine M.; Duderstadt, Karl E.

    2018-03-01

    Single-molecule approaches present a powerful way to obtain detailed kinetic information at the molecular level. However, the identification of small rate changes is often hindered by the considerable noise present in such single-molecule kinetic data. We present a general method to detect such kinetic change points in trajectories of motion of processive single molecules having Gaussian noise, with a minimum number of parameters and without the need of an assumed kinetic model beyond piece-wise linearity of motion. Kinetic change points are detected using a likelihood ratio test in which the probability of no change is compared to the probability of a change occurring, given the experimental noise. A predetermined confidence interval minimizes the occurrence of false detections. Applying the method recursively to all sub-regions of a single molecule trajectory ensures that all kinetic change points are located. The algorithm presented allows rigorous and quantitative determination of kinetic change points in noisy single molecule observations without the need for filtering or binning, which reduce temporal resolution and obscure dynamics. The statistical framework for the approach and implementation details are discussed. The detection power of the algorithm is assessed using simulations with both single kinetic changes and multiple kinetic changes that typically arise in observations of single-molecule DNA-replication reactions. Implementations of the algorithm are provided in ImageJ plugin format written in Java and in the Julia language for numeric computing, with accompanying Jupyter Notebooks to allow reproduction of the analysis presented here.

  4. Relaxation in Thin Polymer Films Mapped across the Film Thickness by Astigmatic Single-Molecule Imaging

    KAUST Repository

    Oba, Tatsuya

    2012-06-19

    We have studied relaxation processes in thin supported films of poly(methyl acrylate) at the temperature corresponding to 13 K above the glass transition by monitoring the reorientation of single perylenediimide molecules doped into the films. The axial position of the dye molecules across the thickness of the film was determined with a resolution of 12 nm by analyzing astigmatic fluorescence images. The average relaxation times of the rotating molecules do not depend on the overall thickness of the film between 20 and 110 nm. The relaxation times also do not show any dependence on the axial position within the films for the film thickness between 70 and 110 nm. In addition to the rotating molecules we observed a fraction of spatially diffusing molecules and completely immobile molecules. These molecules indicate the presence of thin (<5 nm) high-mobility surface layer and low-mobility layer at the interface with the substrate. (Figure presented) © 2012 American Chemical Society.

  5. Electron-vibron coupling effects on electron transport via a single-molecule magnet

    NARCIS (Netherlands)

    McCaskey, A.; Yamamoto, Y.; Warnock, M.; Burzuri, E.; Van der Zant, H.S.J.; Park, K.

    2015-01-01

    We investigate how the electron-vibron coupling influences electron transport via an anisotropic magnetic molecule, such as a single-molecule magnet (SMM) Fe4, by using a model Hamiltonian with parameter values obtained from density-functional theory (DFT). The magnetic anisotropy parameters,

  6. Fully Streched Single DNA Molecules in a Nanofluidic Chip Show Large-Scale Structural Variation

    DEFF Research Database (Denmark)

    Pedersen, Jonas Nyvold; Marie, Rodolphe; Bauer, D. L.

    2013-01-01

    When stretching and imaging DNA molecules in nanofluidic devices, it is important to know the relation between the physical length as measured in the lab and the distance along the contour of the DNA. Here a single DNA molecule longer than 1 Mbp is loaded into a nanofluidic device consisting of two...

  7. Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

    DEFF Research Database (Denmark)

    Marie, Rodolphe; Pedersen, Jonas Nyvold; L. V. Bauer, David

    2013-01-01

    We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratoryon-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known...

  8. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

    Science.gov (United States)

    Neuman, Keir C.; Nagy, Attila

    2012-01-01

    Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. These techniques are described and illustrated with examples highlighting current capabilities and limitations. PMID:18511917

  9. Single molecule charge transport : From a quantum mechanical to a classical description

    NARCIS (Netherlands)

    Kocherzhenko, A.A.; Grozema, F.C.; Siebbeles, L.D.A.

    2010-01-01

    This paper explores charge transport at the single molecule level. The conductive properties of both small organic molecules and conjugated polymers (molecular wires) are considered. In particular, the reasons for the transition from fully coherent to incoherent charge transport and the approaches

  10. A single molecule switch based on two Pd nanocrystals linked by a ...

    Indian Academy of Sciences (India)

    Tunneling spectroscopy measurements have been carried out on a single molecule device formed by two Pd nanocrystals (dia. ∼ 5 nm) electronically coupled by a conducting molecule, dimercaptodiphenylacetylene. The – data, obtained by positioning the tip over a nanocrystal electrode, exhibit negative differential ...

  11. Quantum interference effects at room temperature in OPV-based single-molecule junctions

    NARCIS (Netherlands)

    Arroyo Rodriguez, C.; Frisenda, R.; Moth-Poulsen, K.; Seldenthuis, J.S.; Bjornholm, T.; Van der Zant, H.S.

    2013-01-01

    Interference effects on charge transport through an individual molecule can lead to a notable modulation and suppression on its conductance. In this letter, we report the observation of quantum interference effects occurring at room temperature in single-molecule junctions based on

  12. Unified Model of Dynamic Forced Barrier Crossing in Single Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Friddle, R W

    2007-06-21

    Thermally activated barrier crossing in the presence of an increasing load can reveal kinetic rate constants and energy barrier parameters when repeated over a range of loading rates. Here we derive a model of the mean escape force for all relevant loading rates--the complete force spectrum. Two well-known approximations emerge as limiting cases; one of which confirms predictions that single-barrier spectra should converge to a phenomenological description in the slow loading limit.

  13. Nanoscale and single-molecule interfacial electron transfer

    DEFF Research Database (Denmark)

    Hansen, Allan Glargaard; Wackerbarth, Hainer; Nielsen, Jens Ulrik

    2003-01-01

    Electrochemical science and technology in the 21st century have reached high levels of sophistication. A fundamental quantum mechanical theoretical frame for interfacial electrochemical electron transfer (ET) was introduced by Revaz Dogonadze. This frame has remained for four decades as a basis...... scanning tunneling microscopy (STM) and single-electron tunneling (SET, or Coulomb blockade) in electrochemical. systems directly in aqueous electrolyte solution and at room temperature. We illustrate the new theoretical formalism and its perspectives by recent cases of electrochemical SET, negative...

  14. The elastic theory of a single DNA molecule

    Indian Academy of Sciences (India)

    the other hand, if there is a negative torsional stress, a pulling force as small as 0.3 pN can distort the native structure of DNA considerably [5,6]. Related to the latter, there has been recent progress in understanding the force–extension curves of single-stranded DNA. (ssDNA) and RNA [7–12]. Many distinct transitions have ...

  15. Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

    Science.gov (United States)

    Varela, Juan A.; Dupuis, Julien P.; Etchepare, Laetitia; Espana, Agnès; Cognet, Laurent; Groc, Laurent

    2016-03-01

    Single-molecule imaging has changed the way we understand many biological mechanisms, particularly in neurobiology, by shedding light on intricate molecular events down to the nanoscale. However, current single-molecule studies in neuroscience have been limited to cultured neurons or organotypic slices, leaving as an open question the existence of fast receptor diffusion in intact brain tissue. Here, for the first time, we targeted dopamine receptors in vivo with functionalized quantum dots and were able to perform single-molecule tracking in acute rat brain slices. We propose a novel delocalized and non-inflammatory way of delivering nanoparticles (NPs) in vivo to the brain, which allowed us to label and track genetically engineered surface dopamine receptors in neocortical neurons, revealing inherent behaviour and receptor activity regulations. We thus propose a NP-based platform for single-molecule studies in the living brain, opening new avenues of research in physiological and pathological animal models.

  16. Break junction under electrochemical gating: testbed for single-molecule electronics.

    Science.gov (United States)

    Huang, Cancan; Rudnev, Alexander V; Hong, Wenjing; Wandlowski, Thomas

    2015-02-21

    Molecular electronics aims to construct functional molecular devices at the single-molecule scale. One of the major challenges is to construct a single-molecule junction and to further manipulate the charge transport through the molecular junction. Break junction techniques, including STM break junctions and mechanically controllable break junctions are considered as testbed to investigate and control the charge transport on a single-molecule scale. Moreover, additional electrochemical gating provides a unique opportunity to manipulate the energy alignment and molecular redox processes for a single-molecule junction. In this review, we start from the technical aspects of the break junction technique, then discuss the molecular structure-conductance correlation derived from break junction studies, and, finally, emphasize electrochemical gating as a promising method for the functional molecular devices.

  17. Multicolour single molecule imaging in cells with near infra-red dyes.

    Directory of Open Access Journals (Sweden)

    Christopher J Tynan

    Full Text Available The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging.A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells.We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470-1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.

  18. Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

    Science.gov (United States)

    Tynan, Christopher J.; Clarke, David T.; Coles, Benjamin C.; Rolfe, Daniel J.; Martin-Fernandez, Marisa L.; Webb, Stephen E. D.

    2012-01-01

    Background The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. Methodology/Principal Findings A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells. Conclusions/Significance We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging. PMID:22558412

  19. Isoforms of green fluorescent protein differ from each other in solvent molecules 'trapped' inside this protein.

    Science.gov (United States)

    Glukhova, Kseniya F; Marchenkov, Victor V; Melnik, Tatiana N; Melnik, Bogdan S

    2017-05-01

    Green fluorescent protein (GFP) has been studied quite thoroughly, however, up to now some experimental data have not been explained explicitly. For example, under native conditions this protein can have two isoforms differing in their mobility in gel. In this case, no differences between the isoforms are revealed under denaturing conditions. In order to understand the difference in the isoforms of this protein, we have investigated GFP-cycle3 using mass spectrometry, gel electrophoresis, size exclusion chromatography, microcalorimetry, and spectroscopy methods under varying conditions. We have also designed and studied three mutant forms of this protein with substitutions of amino acid residues inside the GFP barrel. The mutations have allowed us to influence the formation of different GFP isoforms. Each of the mutant proteins has predominantly only one isoform. As a result of the performed research, it can be concluded that most likely the GFP isoforms differ in the solvent molecules 'trapped' inside the GFP barrel. In their turn, these molecules have an effect on the protein charge and consequently on its mobility at electrophoresis under native conditions.

  20. Stretching, twisting and supercoiling in short, single DNA molecules

    Science.gov (United States)

    Lam, Pui-Man; Zhen, Yi

    2018-02-01

    We had combined the Neukirch-Marko model that describes the extension, torque and supercoiling in single, stretched and twisted DNA of infinite contour length, with a form of the free energy suggested by Sinha and Samuels to describe short DNA, with contour length only a few times the persistence length. We find that the free energy of the stretched but untwisted DNA, is significantly modified from its infinitely length value and this in turn modifies significantly the torque and supercoiling. We show that this is consistent with short DNA being more flexible than infinitely long DNA. We hope our results will stimulate experimental investigation of torque and supercoiling in short DNA.

  1. Magnetization relaxation of single molecule magnets after field cooling

    Science.gov (United States)

    Fernandez, Julio F.; Alonso, Juan J.

    2004-03-01

    Magnetic clusters, such as Fe8 and Mn_12, behave at low temperatures as large single spins S. In crystals, anisotropy energies U allow magnetic relaxation only through tunneling at k_BTstackrelspins with dipolar interactions. To mimic tunneling effects, a spin on a lattice site where h is within some tunnel window -h_w

  2. Extracting physics of life at the molecular level: A review of single-molecule data analyses.

    Science.gov (United States)

    Colomb, Warren; Sarkar, Susanta K

    2015-06-01

    Studying individual biomolecules at the single-molecule level has proved very insightful recently. Single-molecule experiments allow us to probe both the equilibrium and nonequilibrium properties as well as make quantitative connections with ensemble experiments and equilibrium thermodynamics. However, it is important to be careful about the analysis of single-molecule data because of the noise present and the lack of theoretical framework for processes far away from equilibrium. Biomolecular motion, whether it is free in solution, on a substrate, or under force, involves thermal fluctuations in varying degrees, which makes the motion noisy. In addition, the noise from the experimental setup makes it even more complex. The details of biologically relevant interactions, conformational dynamics, and activities are hidden in the noisy single-molecule data. As such, extracting biological insights from noisy data is still an active area of research. In this review, we will focus on analyzing both fluorescence-based and force-based single-molecule experiments and gaining biological insights at the single-molecule level. Inherently nonequilibrium nature of biological processes will be highlighted. Simulated trajectories of biomolecular diffusion will be used to compare and validate various analysis techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Structural studies on proton/protonation of the protein molecule

    International Nuclear Information System (INIS)

    Morimoto, Yukio; Kida, Akiko; Chatake, Toshiyuki; Yamaguchi, Hiroshi; Hosokawa, Keiichi; Murakami, Takuto; Umino, Masaaki; Tanaka, Ichiro; Hisatome, Ichiro; Yanagisawa, Yasutake; Fujiwara, Satoshi; Hidaka, Yuji; Shimamoto, Shigeru; Fujiwara, Mitsutoshi; Nakanishi, Takeyoshi

    2015-01-01

    This paper reports three studies involved in the analysis of protons and protonation at physiologically active sites in protein molecules. (1) 'Elucidation of the higher-order structure formation and activity performing mechanism of yeast proteasome.' With an aim to apply to anti-cancer drugs, this study performed the shape analysis of the total structure of 26S proteasome using small-angle X-ray scattering to clarify the complex form where controlling elements bonded to the both ends of 20S catalyst body, and analyzed the complex structure between the active sites of 20S and inhibitor (drug). (2) 'Basic study on the neutron experiment of biomolecules such as physiologically active substances derived from Natto-bacteria.' This study conducted the purification, crystallization, and X-ray analysis experiment of nattokinase; high-grade purification and solution experiment of vitamin K2 (menaquinone-7); and Z-DNA crystal structure study related to the neutron crystal analysis of DNA as another biomolecule structure study. (3) 'Functional evaluation on digestive enzymes derived from Nephila clavata.' As an Alzheimer's disease-related amyloid fibril formation model, this study carried out elucidation on the fibrosis and fiber-forming mechanism of the traction fiber of Nephila clavata, and the functional analysis of its degrading enzyme. (A.O.)

  4. Evaluation of the Electronic Structure of Single-Molecule Junctions Based on Current-Voltage and Thermopower Measurements: Application to C60 Single-Molecule Junction.

    Science.gov (United States)

    Komoto, Yuki; Isshiki, Yuji; Fujii, Shintaro; Nishino, Tomoaki; Kiguchi, Manabu

    2017-02-16

    The electronic structure of molecular junctions has a significant impact on their transport properties. Despite the decisive role of the electronic structure, a complete characterization of the electronic structure remains a challenge. This is because there is no straightforward way of measuring electron spectroscopy for an individual molecule trapped in a nanoscale gap between two metal electrodes. Herein, a comprehensive approach to obtain a detailed description of the electronic structure in single-molecule junctions based on the analysis of current-voltage (I-V) and thermoelectric characteristics is described. It is shown that the electronic structure of the prototypical C 60 single-molecule junction can be resolved by analyzing complementary results of the I-V and thermoelectric measurement. This combined approach confirmed that the C 60 single-molecule junction was highly conductive with molecular electronic conductances of 0.033 and 0.003 G 0 and a molecular Seebeck coefficient of -12 μV K -1 . In addition, we revealed that charge transport was mediated by a LUMO whose energy level was located 0.5≈0.6 eV above the Fermi level of the Au electrode. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Spin transport properties of single metallocene molecules attached to single-walled carbon nanotubes via nickel adatoms

    Science.gov (United States)

    Wei, Peng; Sun, Lili; Benassi, Enrico; Shen, Ziyong; Sanvito, Stefano; Hou, Shimin

    2011-06-01

    The spin-dependent transport properties of single ferrocene, cobaltocene, and nickelocene molecules attached to the sidewall of a (4,4) armchair single-walled carbon nanotube via a Ni adatom are investigated by using a self-consistent ab initio approach that combines the non-equilibrium Green's function formalism with the spin density functional theory. Our calculations show that the Ni adatom not only binds strongly to the sidewall of the nanotube, but also maintains the spin degeneracy and affects little the transmission around the Fermi level. When the Ni adatom further binds to a metallocene molecule, its density of states is modulated by that of the molecule and electron scattering takes place in the nanotube. In particular, we find that for both cobaltocene and nickelocene the transport across the nanotube becomes spin-polarized. This demonstrates that metallocene molecules and carbon nanotubes can become a promising materials platform for applications in molecular spintronics.

  6. Chemical Principles and Interference in the Electrical Conductance of Single Molecules

    DEFF Research Database (Denmark)

    Borges, Anders Christian

    The electrical conductance of single molecules are routinely reported in the scientific literature and off-resonant coherent tunneling is believed to be the mechanism for transport in some of these experiments. In these experiments it is observed that, in spite of similar molecular structures......-Tunneling Microscope Break-Junction experiments (STM-BJ). It is demonstrated that these links can be used to design molecules exhibiting surprising interference effects and to interpret and predict the trends in the characteristic conductance of single molecules without resorting to numerical computational methods...

  7. Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

    Science.gov (United States)

    Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; Zant, Herre S. J. van der

    2015-08-01

    It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the molecule is reduced resulting into a ten-fold increase in the off-resonant differential conductance. Theoretical calculations link the change in differential conductance to a reduction-induced change in conjugation, thereby lifting destructive interference of transport pathways.

  8. Protein Frustratometer 2: a tool to localize energetic frustration in protein molecules, now with electrostatics

    Science.gov (United States)

    Parra, R. Gonzalo; Schafer, Nicholas P.; Radusky, Leandro G.; Tsai, Min-Yeh; Guzovsky, A. Brenda; Wolynes, Peter G.; Ferreiro, Diego U.

    2016-01-01

    The protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins’ biological behavior. It compares the energy distributions of the native state with respect to structural decoys. The network of minimally frustrated interactions encompasses the folding core of the molecule. Sites of high local frustration often correlate with functional regions such as binding sites and regions involved in allosteric transitions. We present here an upgraded version of a webserver that measures local frustration. The new implementation that allows the inclusion of electrostatic energy terms, important to the interactions with nucleic acids, is significantly faster than the previous version enabling the analysis of large macromolecular complexes within a user-friendly interface. The webserver is freely available at URL: http://frustratometer.qb.fcen.uba.ar. PMID:27131359

  9. Full counting statistics of a single-molecule quantum dot

    Science.gov (United States)

    Dong, Bing; Ding, G. H.; Lei, X. L.

    2013-08-01

    We investigate the full counting statistics of a single quantum dot strongly coupled to a local phonon and weakly tunnel connected to two metallic electrodes. By employing the generalized nonequilibrium Green-function method and the Lang-Firsov transformation, we derive an explicit analytical formula for the cumulant generating function, which makes one able to identify distinctly the elastic and inelastic contributions to the current and zero-frequency shot noise. We find that at zero temperature, the inelastic effect causes upward steps in the current and downward jumps in the noise at the bias voltages corresponding to the opening of the inelastic channels, which are ascribed to the vibration-induced complex dependencies of electronic self-energies on the energy and bias voltage. More interestingly, the Fano factor exhibits oscillatory behavior with increasing bias voltage and its minimum value is observed to be smaller than one-half.

  10. Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy

    Directory of Open Access Journals (Sweden)

    Ilko Bald

    2014-09-01

    Full Text Available DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates.

  11. Surface single-molecule dynamics controlled by entropy at low temperatures

    Science.gov (United States)

    Gehrig, J. C.; Penedo, M.; Parschau, M.; Schwenk, J.; Marioni, M. A.; Hudson, E. W.; Hug, H. J.

    2017-02-01

    Configuration transitions of individual molecules and atoms on surfaces are traditionally described using an Arrhenius equation with energy barrier and pre-exponential factor (attempt rate) parameters. Characteristic parameters can vary even for identical systems, and pre-exponential factors sometimes differ by orders of magnitude. Using low-temperature scanning tunnelling microscopy (STM) to measure an individual dibutyl sulfide molecule on Au(111), we show that the differences arise when the relative position of tip apex and molecule changes by a fraction of the molecule size. Altering the tip position on that scale modifies the transition's barrier and attempt rate in a highly correlated fashion, which results in a single-molecular enthalpy-entropy compensation. Conversely, appropriately positioning the STM tip allows selecting the operating point on the compensation line and modifying the transition rates. The results highlight the need to consider entropy in transition rates of single molecules, even at low temperatures.

  12. A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

    Science.gov (United States)

    Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

    Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels

  13. Quantifying and optimizing single-molecule switching nanoscopy at high speeds.

    Directory of Open Access Journals (Sweden)

    Yu Lin

    Full Text Available Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.

  14. Preparation of zinc sulfide nanocrystallites from single-molecule precursors

    Science.gov (United States)

    Palve, Anil M.; Garje, Shivram S.

    2011-07-01

    Zinc sulfide nanocrystallites were prepared using Zinc(II) thiosemicarbazone complexes of the types Zn(L) 2 and ZnCl 2(LH) 2 (where, LH=thiosemicarbazones of cinnamaldehyde, 4-chlorobenzaldehyde, indol-3-carboxaldehyde and thiophene-2-carboxaldehyde) as single source precursors by solvothermal decomposition in ethylene glycol and ethylene diamine in few cases. The materials were characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction, energy dispersive X-ray analysis and UV-vis and IR spectroscopy. Solvothermal decomposition in ethylene glycol resulted in the formation of hexagonal ZnS (JCPDS: 36-1450) as evident from the XRD patterns. However, XRD shows formation of hybrid material, ZnS 0.5EN in case of solvothermal decomposition in ethylenediamine. Infrared spectra authenticate the capping of ethylene glycol and ethylenediamine on ZnS and ZnS 0.5EN, respectively. TEM images showed formation of spherical nanoparticles for the materials obtained from ethylene glycol, whereas plate-like morphology is observed in case of materials obtained from ethylene diamine. The blue shift of absorption bands compared to bands of bulk materials in the UV-vis spectra supports the formation of smaller particles.

  15. Atomic force microscopy and spectroscopy to probe single membrane proteins in lipid bilayers.

    Science.gov (United States)

    Sapra, K Tanuj

    2013-01-01

    The atomic force microscope (AFM) has opened vast avenues hitherto inaccessible to the biological scientist. The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever stylus is aptly termed as a "lab on a tip" owing to its versatility as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples assert that the AFM can be used to study the mechanical properties and monitor processes of single proteins and single cells, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of membrane proteins. Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theoretical, and practical skills.

  16. From isolated light-harvesting complexes to the thylakoid membrane: a single-molecule perspective

    Science.gov (United States)

    Gruber, J. Michael; Malý, Pavel; Krüger, Tjaart P. J.; Grondelle, Rienk van

    2018-01-01

    The conversion of solar radiation to chemical energy in plants and green algae takes place in the thylakoid membrane. This amphiphilic environment hosts a complex arrangement of light-harvesting pigment-protein complexes that absorb light and transfer the excitation energy to photochemically active reaction centers. This efficient light-harvesting capacity is moreover tightly regulated by a photoprotective mechanism called non-photochemical quenching to avoid the stress-induced destruction of the catalytic reaction center. In this review we provide an overview of single-molecule fluorescence measurements on plant light-harvesting complexes (LHCs) of varying sizes with the aim of bridging the gap between the smallest isolated complexes, which have been well-characterized, and the native photosystem. The smallest complexes contain only a small number (10-20) of interacting chlorophylls, while the native photosystem contains dozens of protein subunits and many hundreds of connected pigments. We discuss the functional significance of conformational dynamics, the lipid environment, and the structural arrangement of this fascinating nano-machinery. The described experimental results can be utilized to build mathematical-physical models in a bottom-up approach, which can then be tested on larger in vivo systems. The results also clearly showcase the general property of biological systems to utilize the same system properties for different purposes. In this case it is the regulated conformational flexibility that allows LHCs to switch between efficient light-harvesting and a photoprotective function.

  17. Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

    Science.gov (United States)

    Kuzmenko, Anton; Tankov, Stoyan; English, Brian P.; Tarassov, Ivan; Tenson, Tanel; Kamenski, Piotr; Elf, Johan; Hauryliuk, Vasili

    2011-12-01

    Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.

  18. Packing topology in crystals of proteins and small molecules: a comparison.

    Science.gov (United States)

    Carugo, Oliviero; Blatova, Olga A; Medrish, Elena O; Blatov, Vladislav A; Proserpio, Davide M

    2017-10-16

    We compared the topologies of protein and small molecule crystals, which have many common features - both are molecular crystals with intermolecular interactions much weaker than intramolecular interactions. They also have different features - a considerably large fraction of the volume of protein crystals is occupied by liquid water while no room is available to other molecules in small molecule crystals. We analyzed the overall and local topology and performed multilevel topological analyses (with the software package ToposPro) of carefully selected high quality sets of protein and small molecule crystal structures. Given the suboptimal packing of protein crystals, which is due the special shape and size of proteins, it would be reasonable to expect that the topology of protein crystals is different from the topology of small molecule crystals. Surprisingly, we discovered that these two types of crystalline compounds have strikingly similar topologies. This might suggest that molecular crystal formations share symmetry rules independent of molecular dimension.

  19. Development of new photon-counting detectors for single-molecule fluorescence microscopy

    Science.gov (United States)

    Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

    2013-01-01

    Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185

  20. Supramolecular Systems and Chemical Reactions in Single-Molecule Break Junctions.

    Science.gov (United States)

    Li, Xiaohui; Hu, Duan; Tan, Zhibing; Bai, Jie; Xiao, Zongyuan; Yang, Yang; Shi, Jia; Hong, Wenjing

    2017-04-01

    The major challenges of molecular electronics are the understanding and manipulation of the electron transport through the single-molecule junction. With the single-molecule break junction techniques, including scanning tunneling microscope break junction technique and mechanically controllable break junction technique, the charge transport through various single-molecule and supramolecular junctions has been studied during the dynamic fabrication and continuous characterization of molecular junctions. This review starts from the charge transport characterization of supramolecular junctions through a variety of noncovalent interactions, such as hydrogen bond, π-π interaction, and electrostatic force. We further review the recent progress in constructing highly conductive molecular junctions via chemical reactions, the response of molecular junctions to external stimuli, as well as the application of break junction techniques in controlling and monitoring chemical reactions in situ. We suggest that beyond the measurement of single molecular conductance, the single-molecule break junction techniques provide a promising access to study molecular assembly and chemical reactions at the single-molecule scale.

  1. Ninth international conference on hole burning, single molecule and related spectroscopies: science and applications (HBSM 2006)

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2006-07-01

    This conference was organized around 9 sessions: -) single molecule, -) quantum optics, -) hole-burning materials and mechanisms, -) single nano-particle spectroscopy, -) dephasing and spectral diffusion, -) microwave photonics, -) biological systems, -) rare earth doped materials, -) novel laser sources. This document gathers only the slides of the presentations.

  2. Ninth international conference on hole burning, single molecule and related spectroscopies: science and applications (HBSM 2006)

    International Nuclear Information System (INIS)

    2006-01-01

    This conference was organized around 9 sessions: -) single molecule, -) quantum optics, -) hole-burning materials and mechanisms, -) single nano-particle spectroscopy, -) dephasing and spectral diffusion, -) microwave photonics, -) biological systems, -) rare earth doped materials, -) novel laser sources. This document gathers only the slides of the presentations

  3. Nano- and micro-fabrication for single-molecule biological studies

    NARCIS (Netherlands)

    Huang, Z.

    2012-01-01

    Heterogeneity is a general feature in biological system. In order to avoid possible misleading effects of ensemble averaging, and to ensure a correct understanding of the biological system, it is very important to look into individuals, such as a single bio-molecule or a single cell, for details.

  4. Radiation- and phonon-bottleneck--induced tunneling in the Fe8 single-molecule magnet

    Science.gov (United States)

    Bal, M.; Friedman, Jonathan R.; Chen, W.; Tuominen, M. T.; Beedle, C. C.; Rumberger, E. M.; Hendrickson, D. N.

    2008-04-01

    We measure magnetization changes in a single crystal of the single-molecule magnet Fe8 when exposed to intense, short (spin dynamics, allowing observation of thermally assisted resonant tunneling between spin states at the 100 ns time scale. Detailed numerical simulations quantitatively reproduce the data and yield a spin-phonon relaxation time T1~40 ns.

  5. Spectral multitude and spectral dynamics reflect changing conjugation length in single molecules of oligophenylenevinylenes

    KAUST Repository

    Kobayashi, Hiroyuki

    2012-01-01

    Single-molecule study of phenylenevinylene oligomers revealed distinct spectral forms due to different conjugation lengths which are determined by torsional defects. Large spectral jumps between different spectral forms were ascribed to torsional flips of a single phenylene ring. These spectral changes reflect the dynamic nature of electron delocalization in oligophenylenevinylenes and enable estimation of the phenylene torsional barriers. © 2012 The Owner Societies.

  6. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    Science.gov (United States)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  7. Metal-Controlled Magnetoresistance at Room Temperature in Single-Molecule Devices.

    Science.gov (United States)

    Aragonès, Albert C; Aravena, Daniel; Valverde-Muñoz, Francisco J; Real, José Antonio; Sanz, Fausto; Díez-Pérez, Ismael; Ruiz, Eliseo

    2017-04-26

    The appropriate choice of the transition metal complex and metal surface electronic structure opens the possibility to control the spin of the charge carriers through the resulting hybrid molecule/metal spinterface in a single-molecule electrical contact at room temperature. The single-molecule conductance of a Au/molecule/Ni junction can be switched by flipping the magnetization direction of the ferromagnetic electrode. The requirements of the molecule include not just the presence of unpaired electrons: the electronic configuration of the metal center has to provide occupied or empty orbitals that strongly interact with the junction metal electrodes and that are close in energy to their Fermi levels for one of the electronic spins only. The key ingredient for the metal surface is to provide an efficient spin texture induced by the spin-orbit coupling in the topological surface states that results in an efficient spin-dependent interaction with the orbitals of the molecule. The strong magnetoresistance effect found in this kind of single-molecule wire opens a new approach for the design of room-temperature nanoscale devices based on spin-polarized currents controlled at molecular level.

  8. Drift correction for single-molecule imaging by molecular constraint field, a distance minimum metric

    International Nuclear Information System (INIS)

    Han, Renmin; Wang, Liansan; Xu, Fan; Zhang, Yongdeng; Zhang, Mingshu; Liu, Zhiyong; Ren, Fei; Zhang, Fa

    2015-01-01

    The recent developments of far-field optical microscopy (single molecule imaging techniques) have overcome the diffraction barrier of light and improve image resolution by a factor of ten compared with conventional light microscopy. These techniques utilize the stochastic switching of probe molecules to overcome the diffraction limit and determine the precise localizations of molecules, which often requires a long image acquisition time. However, long acquisition times increase the risk of sample drift. In the case of high resolution microscopy, sample drift would decrease the image resolution. In this paper, we propose a novel metric based on the distance between molecules to solve the drift correction. The proposed metric directly uses the position information of molecules to estimate the frame drift. We also designed an algorithm to implement the metric for the general application of drift correction. There are two advantages of our method: First, because our method does not require space binning of positions of molecules but directly operates on the positions, it is more natural for single molecule imaging techniques. Second, our method can estimate drift with a small number of positions in each temporal bin, which may extend its potential application. The effectiveness of our method has been demonstrated by both simulated data and experiments on single molecular images

  9. Assembly and diploid architecture of an individual human genome via single-molecule technologies.

    Science.gov (United States)

    Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

    2015-08-01

    We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

  10. Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

    Science.gov (United States)

    Kaplan, Andrew; Morquette, Barbara; Kroner, Antje; Leong, SooYuen; Madwar, Carolin; Sanz, Ricardo; Banerjee, Sara L; Antel, Jack; Bisson, Nicolas; David, Samuel; Fournier, Alyson E

    2017-03-08

    Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Role of ICAM-1 polymorphisms (G241R, K469E) in mediating its single-molecule binding ability: Atomic force microscopy measurements on living cells

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Rui [Chinese (301) General Hospital, 28 Fuxing Road, Haidian District, Beijing 100853 (China); Yi, Shaoqiong [Beijing Institute of Biotechnology, 20 Dongdajie, Fengtai, Beijing 100071 (China); Zhang, Xuejie [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry Chinese Academy of Sciences, 2 Zhongguancun North 1st Street, Beijing 100190 (China); Liu, Huiliang, E-mail: lhl518@vip.sina.com [Department of Cardiology, The General Hospital of Chinese People’s Armed Police Forces, Beijing 100039 (China); Fang, Xiaohong, E-mail: xfang@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry Chinese Academy of Sciences, 2 Zhongguancun North 1st Street, Beijing 100190 (China)

    2014-06-13

    Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.

  12. Nonequilibrium Chemical Effects in Single-Molecule SERS Revealed by Ab Initio Molecular Dynamics Simulations

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Sean A.; Apra, Edoardo; Govind, Niranjan; Hess, Wayne P.; El-Khoury, Patrick Z.

    2017-02-03

    Recent developments in nanophotonics have paved the way for achieving significant advances in the realm of single molecule chemical detection, imaging, and dynamics. In particular, surface-enhanced Raman scattering (SERS) is a powerful analytical technique that is now routinely used to identify the chemical identity of single molecules. Understanding how nanoscale physical and chemical processes affect single molecule SERS spectra and selection rules is a challenging task, and is still actively debated. Herein, we explore underappreciated chemical phenomena in ultrasensitive SERS. We observe a fluctuating excited electronic state manifold, governed by the conformational dynamics of a molecule (4,4’-dimercaptostilbene, DMS) interacting with a metallic cluster (Ag20). This affects our simulated single molecule SERS spectra; the time trajectories of a molecule interacting with its unique local environment dictates the relative intensities of the observable Raman-active vibrational states. Ab initio molecular dynamics of a model Ag20-DMS system are used to illustrate both concepts in light of recent experimental results.

  13. Single-molecule chemical reaction reveals molecular reaction kinetics and dynamics.

    Science.gov (United States)

    Zhang, Yuwei; Song, Ping; Fu, Qiang; Ruan, Mingbo; Xu, Weilin

    2014-06-25

    Understanding the microscopic elementary process of chemical reactions, especially in condensed phase, is highly desirable for improvement of efficiencies in industrial chemical processes. Here we show an approach to gaining new insights into elementary reactions in condensed phase by combining quantum chemical calculations with a single-molecule analysis. Elementary chemical reactions in liquid-phase, revealed from quantum chemical calculations, are studied by tracking the fluorescence of single dye molecules undergoing a reversible redox process. Statistical analyses of single-molecule trajectories reveal molecular reaction kinetics and dynamics of elementary reactions. The reactivity dynamic fluctuations of single molecules are evidenced and probably arise from either or both of the low-frequency approach of the molecule to the internal surface of the SiO2 nanosphere or the molecule diffusion-induced memory effect. This new approach could be applied to other chemical reactions in liquid phase to gain more insight into their molecular reaction kinetics and the dynamics of elementary steps.

  14. Electronic Transport in Single Molecule Junctions: Control of the Molecule-Electrode Coupling Through Intramolecular Tunneling Barriers

    DEFF Research Database (Denmark)

    Danilov, Andrey; Kubatkin, Sergey; Kafanov, Sergey

    2008-01-01

    We report on single molecule electron transport measurements of two oligophenylenevinylene (OPV3) derivatives placed in a nanogap between gold (Au) or lead (Pb) electrodes in a field effect transistor device. Both derivatives contain thiol end groups that allow chemical binding to the electrodes....... One derivative has additional methylene groups separating the thiols from the delocalized -electron system. The insertion of methylene groups changes the open state conductance by 3-4 orders of magnitude and changes the transport mechanism from a coherent regime with finite zero-bias conductance...

  15. Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single-Molecule Tracking Study.

    Science.gov (United States)

    Möckl, Leonhard; Lindhorst, Thisbe K; Bräuchle, Christoph

    2016-03-16

    We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Vesicle Encapsulation Studies Reveal that Single Molecule Ribozyme Heterogeneities Are Intrinsic

    Science.gov (United States)

    Okumus, Burak; Wilson, Timothy J.; Lilley, David M. J.; Ha, Taekjip

    2004-01-01

    Single-molecule measurements have revealed that what were assumed to be identical molecules can differ significantly in their static and dynamic properties. One of the most striking examples is the hairpin ribozyme, which was shown to exhibit two to three orders of magnitude variation in folding kinetics between molecules. Although averaged behavior of single molecules matched the bulk solution data, it was not possible to exclude rigorously the possibility that the variations around the mean values arose from different ways of interacting with the surface environment. To test this, we minimized the molecules' interaction with the surface by encapsulating DNA or RNA molecules inside 100- to 200-nm diameter unilamellar vesicles, following the procedures described by Haran and coworkers. Vesicles were immobilized on a supported lipid bilayer via biotin-streptavidin linkages. We observed no direct binding of DNA or RNA on the supported bilayer even at concentrations exceeding 100 nM, indicating that these molecules do not bind stably on the membrane. Since the vesicle diameter is smaller than the resolution of optical microscopy, the lateral mobility of the molecules is severely constrained, allowing long observation periods. We used fluorescence correlation spectroscopy, nuclease digestion, and external buffer exchange to show that the molecules were indeed encapsulated within the vesicles. When contained within vesicles, the natural form of the hairpin ribozyme exhibited 50-fold variation in both folding and unfolding rates in 0.5 mM Mg2+, which is identical to what was observed from the molecules tethered directly on the surface. This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules. PMID:15454471

  17. Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

    Science.gov (United States)

    Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

    2018-01-01

    Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

  18. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

  19. madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy

    Science.gov (United States)

    Yi, Jason; Manna, Asit; Barr, Valarie A.; Hong, Jennifer; Neuman, Keir C.; Samelson, Lawrence E.

    2016-01-01

    Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM. PMID:27708141

  20. Integrated Transmission Electron and Single-Molecule Fluorescence Microscopy Correlates Reactivity with Ultrastructure in a Single Catalyst Particle

    OpenAIRE

    Hendriks, Frank C.; Mohammadian, Sajjad; Ristanovic, Zoran; Kalirai, Samanbir; Meirer, Florian; Vogt, Eelco T. C.; Bruijnincx, Pieter C. A.; Gerritsen, Hans; Weckhuysen, Bert M.

    2018-01-01

    Establishing structure–activity relationships in complex, hierarchically structured nanomaterials, such as fluid catalytic cracking (FCC) catalysts, requires characterization with complementary, correlated analysis techniques. An integrated setup has been developed to perform transmission electron microscopy (TEM) and single-molecule fluorescence (SMF) microscopy on such nanostructured samples. Correlated structure–reactivity information was obtained for 100 nm thin, microtomed sections of a ...